MODIDIFED iRNA  AGENTS

ABSTRACT

The invention relates to iRNA agents, which preferably include a monomer in which the ribose moiety has been replaced by a moiety other than ribose. The inclusion of such a monomer can allow for modulation of a property of the iRNA agent into which it is incorporated, e.g., by using the non-ribose moiety as a point to which a ligand or other entity, e.g., a lipophilic moiety. e.g., cholesterol, is directly, or indirectly, tethered. The invention also relates to methods of making and using such modified iRNA agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/849,003, filed Mar. 22, 2013, which is a continuation of U.S.application Ser. No. 12/714,298, filed Feb. 26, 2010, which is acontinuation of U.S. application Ser. No. 10/916,185, filed Aug. 10,2004, now U.S. Pat. No. 7,745,608, which is a continuation-in-part ofInternational Application No. PCT/US2004/011829, filed on Apr. 16, 2004,which claims the benefit of U.S. Provisional Application No. 60/493,986,filed on Aug. 8, 2003; U.S. Provisional Application No. 60/494,597,filed on Aug. 11, 2003; U.S. Provisional Application No. 60/506,341,filed on Sep. 26, 2003; United States Provisional Application No.:

60/518,453, filed on Nov. 7, 2003; U.S. Provisional Application No.60/463, 772, filed on Apr. 17, 2003; U.S. Provisional Application No.60/465,802, filed on Apr. 25, 2003; U.S. Provisional Application No.60/469,612, filed on May 9, 2003; U.S. Provisional Application No.60/510,246, filed on Oct. 9, 2003; U.S. Provisional Application No.60/510,318, filed on Oct. 10, 2003; U.S. Provisional Application No.60/503,414, filed on Sep. 15, 2003; U.S. Provisional Application No.60/465,665, filed on Apr. 25, 2003; International Application No.:PCT/US04/07070, filed on Mar. 8, 2004; International Application No.:PCT/US2004/10586, filed on Apr. 5, 2004; International Application No.:PCT/US2004/11255, filed on Apr. 9, 2004; and International ApplicationNo.: PCT/US2004/011822, filed on Apr. 16, 2004. The contents of all ofthese prior applications are hereby incorporated by reference in theirentireties.

TECHNICAL FIELD

The invention relates to iRNA agents, which preferably include a monomerin which the ribose moiety has been replaced by a moiety other thanribose. The inclusion of such a monomer can allow for modulation of aproperty of the iRNA agent into which it is incorporated, e.g., by usingthe non-ribose moiety as a point to which a ligand or other entity,e.g., a lipophilic moiety. e.g., cholesterol, is directly, orindirectly, tethered. The invention also relates to methods of makingand using such modified iRNA agents.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression when it is introduced into worms (Fire et al.(1998) Nature 391, 806-811). Short dsRNA directs gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and has provided a new tool for studying gene function. RNAi may involvemRNA degradation.

SUMMARY

The inventor has discovered, inter alia, that the ribose sugar of one ormore ribonucleotide subunits of an iRNA agent can be replaced withanother moiety, e.g., a non-carbohydrate (preferably cyclic) carrier. Aribonucleotide subunit in which the ribose sugar of the subunit has beenso replaced is referred to herein as a ribose replacement modificationsubunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e.,all ring atoms are carbon atoms, or a heterocyclic ring system, i.e.,one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen,sulfur. The cyclic carrier may be a monocyclic ring system, or maycontain two or more rings, e.g. fused rings. The cyclic carrier may be afully saturated ring system, or it may contain one or more double bonds.

The carriers further include (i) at least two “backbone attachmentpoints” and (ii) at least one “tethering attachment point.” A “backboneattachment point” as used herein refers to a functional group, e.g. ahydroxyl group, or generally, a bond available for, and that is suitablefor incorporation of the carrier into the backbone, e.g., the phosphate,or modified phosphate, e.g., sulfur containing, backbone, of aribonucleic acid. A “tethering attachment point” in some embodimentsrefers to a constituent ring atom of the cyclic carrier, e.g., a carbonatom or a heteroatom (distinct from an atom which provides a backboneattachment point), that connects a selected moiety. The moiety can be,e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety whichalters a physical property. One of the most preferred moieties is amoiety which promotes entry into a cell, e.g., a lipophilic moiety,e.g., cholesterol. While not wishing to be bound by theory it isbelieved the attachment of a lipohilic agent increases the lipophilicityof an iRNA agent. Optionally, the selected moiety is connected by anintervening tether to the cyclic carrier. Thus, it will often include afunctional group, e.g., an amino group, or generally, provide a bond,that is suitable for incorporation or tethering of another chemicalentity, e.g., a ligand to the constituent ring.

Incorporation of one or more RRMSs described herein into an RNA agent,e.g., an iRNA agent, particularly when tethered to an appropriateentity, can confer one or more new properties to the RNA agent and/oralter, enhance or modulate one or more existing properties in the RNAmolecule. E.g., it can alter one or more of lipophilicity or nucleaseresistance. Incorporation of one or more RRMSs described herein into aniRNA agent can, particularly when the RRMS is tethered to an appropriateentity, modulate, e.g., increase, binding affinity of an iRNA agent to atarget mRNA, change the geometry of the duplex form of the iRNA agent,alter distribution or target the iRNA agent to a particular part of thebody, or modify the interaction with nucleic acid binding proteins(e.g., during RISC formation and strand separation).

Accordingly, in one aspect, the invention features, an iRNA agentpreferably comprising a first strand and a second strand, wherein atleast one subunit having a formula (I) is incorporated into at least oneof said strands:

wherein:

X is N(CO)R⁷, NR⁷ or CH₂;

Y is NR⁸, O, S, CR⁹R¹⁰, or absent;

Z is CR¹¹R¹² or absent;

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^(a),OR^(b), (CH₂)_(n)OR^(a), or (CH₂)_(n)OR^(b), provided that at least oneof R¹, R², R³, R⁴, R⁹, and R¹⁰ is OR^(a) or OR^(b) and that at least oneof R¹, R², R³, R⁴, R⁹, and R¹⁰ is (CH₂)_(n)OR^(a), or (CH₂)_(n)OR^(b)(when the RRMS is terminal, one of R¹, R², R³, R⁴, R⁹, and R¹⁰ willinclude R^(a) and one will include R^(b); when the RRMSS is internal,two of R¹, R², R³, R⁴, R⁹, and R¹⁰ will each include an R^(b));furtherprovided that preferably OR^(a) may only be present with (CH₂)_(n)OR^(b)and (CH₂)_(n)OR^(a) may only be present with OR^(b).

Each of R⁵, R⁶, R¹¹, and R¹² is, independently, H, C₁-C₆ alkyloptionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹ togetherare C₃-C₈ cycloalkyl optionally substituted with R¹⁴;

R⁷ can be a ligand, e.g., R⁷ can be R^(d), or R⁷ can be a ligandtethered indirectly to the carrier, e.g., through a tethering moiety,e.g., C₁-C₂₀ alkyl substituted with NR^(c)R^(d); or C₁-C₂₀ alkylsubstituted with NHC(O)R^(d);

R⁸ is C₁-C₆ alkyl;

R¹³ is hydroxy, C₁-C₄ alkoxy, or halo;

R¹⁴ is NR^(c)R⁷;

R^(a) is H or:

R^(b) is H or:

Each of A and C is, independently, O or S;

B is OH, O⁻, or

R^(c) is H or C₁-C₆ alkyl;

R^(d) is H or a ligand, e.g., a lipophilic ligand, e.g., cholesterol;and

n is 1-4.

Embodiments can include one or more of the following features.

The iRNA agent can be 21 nucleotides in length and there can be a duplexregion of about 19 pairs.

The iRNA agent can include a duplex region between 17 and 23 pairs inlength.

R¹ can be CH₂OR^(a) and R³ can be OR^(b); or R¹ can be CH₂OR^(a) and R⁹can be OR^(b); or R¹ can be CH₂OR^(a) and R² can be OR^(b).

R¹ can be CH₂OR^(b) and R³ can be OR^(b); or R¹ can be CH₂OR^(b) and R⁹can be OR^(b); or R¹ can be CH₂OR^(b) and R² can be OR^(b); or R¹ can beCH₂OR^(b) and R³ can be OR^(a); or R¹ can be CH₂OR^(b) and R⁹ can beOR^(a); or R¹ can be CH₂OR^(b) and R² can be OR^(a).

R¹ can be OR^(a) and R³ can be CH₂OR^(b); or R¹ can be OR^(a) and R⁹ canbe CH₂OR^(b); or R¹ can be OR^(a) and R² can be CH₂OR^(b).

R¹ can be OR^(b) and R³ can be CH₂OR^(b); or R¹ can be OR^(b) and R⁹ canbe CH₂OR^(b); or R¹ can be OR^(b) and R² can be CH₂OR^(b); or R¹ can beOR^(b) and R³ can be CH₂OR^(a); or R¹ can be OR^(b) and R⁹ can beCH₂OR^(a); or R¹ can be OR^(b) and R² can be CH₂OR^(a).

R³ can be CH₂OR^(a) and R⁹ can be OR^(b); or R³ can be CH₂OR^(a) and R⁴can be OR^(b).

R³ can be CH₂OR^(b) and R⁹ can be OR^(b); or R³ can be CH₂OR^(b) and R⁴can be OR^(b); or R³ can be CH₂OR^(b) and R⁹ can be OR^(a); or R³ can beCH₂OR^(b) and R⁴ can be OR^(a).

R³ can be OR^(b) and R⁹ can be CH₂OR^(a); or R³ can be OR^(b) and R⁴ canbe CH₂OR^(a); or R³ can be OR^(b) and R⁹ can be CH₂OR^(b); or R³ can beOR^(b) and R⁴ can be CH₂OR^(b).

R³ can be OR^(a) and R⁹ can be CH₂OR^(b); or R³ can be OR^(a) and R⁴ canbe CH₂OR^(b).

R⁹ can be CH₂OR^(a) and R¹⁰ can be OR^(b).

R⁹ can be CH₂OR^(b) and R¹⁰ can be OR^(b); or R⁹ can be CH₂OR^(b) andR¹⁰ can be OR^(a).

In a preferred embodiment the ribose is replaced with a pyrrolinescaffold or with a 4-hydroxyproline-derived scaffold, and X is N(CO)R⁷or NR⁷, Y is CR⁹R¹⁰, and Z is absent.

R¹ and R³ can be cis or R¹ and R³ can be trans.

n can be 1.

A can be O or S.

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(b); or R¹ can be(CH₂)_(n)OR^(a) and R³ can be OR^(b).

R⁷ can be (CH₂)₅NHR^(d) or (CH₂)₅NHR^(d). R^(d) can be chosen from afolic acid radical; a cholesterol radical; a carbohydrate radical; avitamin A radical; a vitamin E radical; a vitamin K radical. Preferably,R^(d) is a cholesterol radical.

R¹ can be OR^(b) and R³ can be (CH₂)_(n)OR^(b); or R¹ can be OR^(b) andR³ can be (CH₂)_(n)OR^(a); or

R¹ can be OR^(a) and R³ can be (CH₂)_(n)OR^(b); or R¹ can be(CH₂)_(n)OR^(b) and R⁹ can be OR^(a).

R¹ and R⁹ can be cis or R¹ and R⁹ can be trans. R¹ can be OR^(a) and R⁹can be (CH₂)_(n)OR^(b); or R¹ can be (CH₂)_(n)OR^(b) and R⁹ can beOR^(b); or R¹ can be (CH₂)_(n)OR^(a) and R⁹ can be OR^(b); or R¹ can beOR^(b) and R⁹ can be (CH₂)_(n)OR^(b); or R¹ can be OR^(b) and R⁹ can be(CH₂)_(n)OR^(a).

R³ can be (CH₂)_(n)OR^(b) and R⁹ can be OR^(b); or R³ can be(CH₂)_(n)OR^(b) and R⁹ can be OR^(b); or

R³ can be (CH₂)_(n)OR^(a) and R⁹ can be OR^(b); or R³ can be OR^(a) andR⁹ can be (CH₂)_(n)OR^(b); R³ can be OR^(b) and R⁹ can be(CH₂)_(n)OR^(b); or R³ can be OR^(b) and R⁹ can be (CH₂)_(n)OR^(a).

R³ and R⁹ can be cis or R³ and R⁹ can be trans.

In other preferred embodiments the ribose is replaced with a piperidinescaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹².

R⁹ can be (CH₂)_(n)OR^(b) and R¹⁰ can be OR^(a).

n can be 1 or 2.

R⁹ can be (CH₂)_(n)OR^(b) and R¹⁰ can be OR^(b); or R⁹ can be(CH₂)_(n)OR^(a) and R¹⁰ can be OR^(b).

A can be O or S.

R⁷ can be (CH₂)₅NHR^(d) or (CH₂)₅NHR^(d). R^(d) can be selected from afolic acid radical; a cholesterol radical; a carbohydrate radical; avitamin A radical; a vitamin E radical; a vitamin K radical. Preferably,R^(d) is a cholesterol radical.

R³ can be (CH₂)_(n)OR^(b) and R⁴ can be OR^(a); or R³ can be(CH₂)_(n)OR^(b) and R⁴ can be OR^(b); or

R³ can be (CH₂)_(n)OR^(a) and R⁴ can be OR^(b).

R¹ can be (CH₂)_(n)OR^(b) and R² can be OR^(b); or R¹ can be(CH₂)_(n)OR^(b) and R² can be OR^(b); or

R¹ can be (CH₂)_(n)OR^(b) and R² can be OR^(b).

R³ can be (CH₂)_(n)OR^(b) and R⁹ can be OR^(a).

R³ and R⁹ can be cis, or R³ and R⁹ can be trans.

R³ can be (CH₂)_(n)OR^(b) and R⁹ can be OR^(b); or R³ can be(CH₂)_(n)OR^(b) and R⁹ can be ORa; or

R³ can be (CH₂)_(n)OR^(b) and R⁹ can be OR^(b).

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(a).

R¹ and R³ can be cis, or R¹ and R³ can be trans. R³ can be ORa and R⁹can be (CH₂)_(n)OR^(b).

R¹ can be ORa and R³ can be (CH₂)_(n)OR^(b).

In other preferred embodiments the ribose is replaced with a piperazinescaffold, and X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹².

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(a).

R¹ and R³ can be cis or R¹ and R³ can be trans.

n can be 1.

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(b); or R¹ can be(CH₂)_(n)OR^(a) and R³ can be OR^(b).

A can be O or S, preferably S.

R⁷ can be (CH₂)₅NHR^(d) or (CH₂)₅NHR^(d). R^(d) can be chosen from thegroup of a folic acid radical; a cholesterol radical; a carbohydrateradical; a vitamin A radical; a vitamin E radical; a vitamin K radical.Preferably, R^(d) is a cholesterol radical.

R⁸ can be CH₃.

R¹ can be OR^(a) and R³ can be (CH₂)_(n)OR^(b).

In other preferred embodiments the ribose is replaced with a morpholinoscaffold, and X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹².

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(a).

R¹ and R³ can be cis, or R¹ and R³ can be trans.

n can be 1.

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(b); of R¹ can be(CH₂)_(n)OR^(b) and R³ can be OR^(b).

A can be O or S.

R⁷ can be (CH₂)₅NHR^(d) or (CH₂)₅NHR^(d). R^(d) can be chosen from thegroup of a folic acid radical; a cholesterol radical; a carbohydrateradical; a vitamin A radical; a vitamin E radical; a vitamin K radical.Preferably, R^(d) is a cholesterol radical.

R⁸ can be CH₃.

R¹ can be OR^(a) and R³ can be (CH₂)_(n)OR^(b).

In other preferred embodiments the ribose is replaced with a decalinscaffold, and X is CH₂; Y is CR⁹R¹⁰; and Z is CR¹¹R¹²; and R⁵ and R¹¹together are C⁶ cycloalkyl.

R⁶ can be C(O)NHR⁷.

R¹² can be hydrogen.

R⁶ and R¹² can be trans.

R³ can be OR^(a) and R⁹ can be (CH₂)_(n)OR^(b).

R³ and R⁹ can be cis, or R³ and R⁹ can be trans.

n can be 1 or 2.

R³ can be OR^(b) and R⁹ can be (CH₂)_(n)OR^(b); or R³ can be OR^(b) andR⁹ can be (CH₂)_(n)OR^(a).

A can be O or S.

R⁷ can be (CH₂)₅NHR^(d) or (CH₂)₅NHR^(d). R^(d) can be chosen from thegroup of a folic acid radical; a cholesterol radical; a carbohydrateradical; a vitamin A radical; a vitamin E radical; a vitamin K radical.Preferably, R^(d) is a cholesterol radical.

In other preferred embodiments the ribose is replaced with adecalin/indane scafold, e.g., X is CH₂; Y is CR⁹R¹⁰; and Z is CR¹¹R¹²;and R⁵ and R¹¹ together are C⁵ cycloalkyl.

R⁶ can be CH₃.

R¹² can be hydrogen.

R⁶ and R¹² can be trans.

R³ can be OR^(a) and R⁹ can be (CH₂)_(n)OR^(b).

R³ and R⁹ can be cis, or R³ and R⁹ can be trans.

n can be 1 or 2.

R³ can be OR^(b) and R⁹ can be (CH₂)_(n)OR^(a); or R³ can be OR^(b) andR⁹ can be (CH₂)_(n)OR^(a).

A can be O or S.

R¹⁴ can be N(CH3)R⁷. R⁷ can be (CH₂)₅NHR^(d) or (CH₂)₅NHR^(d). R^(d) canbe chosen from the group of a folic acid radical; a cholesterol radical;a carbohydrate radical; a vitamin A radical; a vitamin E radical; avitamin K radical. Preferably, R^(d) is a cholesterol radical.

In another aspect, this invention features an iRNA agent comprising afirst strand and a second strand, wherein at least one one subunithaving a formula (II) is incorporated into at least one of said strands:

X is N(CO)R⁷ or NR⁷;

Each of R¹ and R² is, independently, OR^(a), OR^(b), (CH₂)_(n)OR^(a), or(CH₂)_(n)OR^(b), provided that one of R¹ and R² is OR^(a) or OR^(b) andthe other is (CH₂)_(n)OR^(a) or (CH₂)_(n)OR^(b) (when the RRMS isterminal, one of R¹ or R² will include R^(a) and one will include R^(b);when the RRMSS is internal, both R¹ and R² will each include anR^(b));further provided that preferably OR^(a) may only be present with(CH₂)_(n)OR^(b) and (CH₂)_(n)OR^(a) may only be present with OR^(b);

R⁷ is C₁-C₂₀ alkyl substituted with NR^(c)R^(d);

R⁸ is C₁-C₆ alkyl;

R¹³ is hydroxy, C₁-C₄ alkoxy, or halo;

R¹⁴ is NR^(c)R⁷;

R^(a) is:

R^(b) is

Each of A and C is, independently, O or S;

B is OH, O⁻, or

R^(c) is H or C₁-C₆ alkyl;

R^(d) is H or a ligand; and

n is 1-4.

Embodiments can include one or more of the features described above.

In a further aspect, this invention features an iRNA agent having afirst strand and a second strand, wherein at least one subunit having aformula (I) or formula (II) is incorporated into at least one of saidstrands.

In one aspect, this invention features an iRNA agent having a firststrand and a second strand, wherein at least two subunits having aformula (I) and/or formula (II) are incorporated into at least one ofsaid strands.

In another aspect, this invention provides a method of making an iRNAagent described herein having a first strand and a second strand inwhich at least one subunit of formula (I) and/or (II) is incorporated inthe strands. The method includes contacting the first strand with thesecond strand.

In a further aspect, this invention provides a method of modulatingexpression of a target gene, the method includes administering an iRNAagent described herein having a first strand and a second strand inwhich at least one subunit of formula (I) and/or (II) is incorporated inthe strands. to a subject.

In one aspect, this invention features a pharmaceutical compositionhaving an iRNA agent described herein having a first strand and a secondstrand in which at least one subunit of formula (I) and/or (II) isincorporated in the strands and a pharmaceutically acceptable carrier.

RRMSs described herein may be incorporated into any double-strandedRNA-like molecule described herein, e.g., an iRNA agent. An iRNA agentmay include a duplex comprising a hybridized sense and antisense strand,in which the antisense strand and/or the sense strand may include one ormore of the RRMSs described herein. An RRMS can be introduced at one ormore points in one or both strands of a double-stranded iRNA agent. AnRRMS can be placed at or near (within 1, 2, or 3 positions) of the 3′ or5′ end of the sense strand or at near (within 2 or 3 positions of) the3′ end of the antisense strand. In some embodiments it is preferred tonot have an RRMS at or near (within 1, 2, or 3 positions of) the 5′ endof the antisense strand. An RRMS can be internal, and will preferably bepositioned in regions not critical for antisense binding to the target.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or3 positions of) the 3′ end of the antisense strand. In an embodiment, aniRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the3′ end of the antisense strand and at (or within 1, 2, or 3 positionsof) the 3′ end of the sense strand. In an embodiment, an iRNA agent mayhave an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of theantisense strand and an RRMS at the 5′ end of the sense strand, in whichboth ligands are located at the same end of the iRNA agent.

In certain embodiments, two ligands are tethered, preferably, one oneach strand and are hydrophobic moieties. While not wishing to be boundby theory, it is believed that pairing of the hydrophobic ligands canstabilize the iRNA agent via intermolecular van der Waals interactions.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′end of the sense strand, in which both RRMSs may share the same ligand(e.g., cholic acid) via connection of their individual tethers toseparate positions on the ligand. A ligand shared between two proximalRRMSs is referred to herein as a “hairpin ligand.”

In other embodiments, an iRNA agent may have an RRMS at the 3′ end ofthe sense strand and an RRMS at an internal position of the sensestrand. An iRNA agent may have an RRMS at an internal position of thesense strand; or may have an RRMS at an internal position of theantisense strand; or may have an RRMS at an internal position of thesense strand and an RRMS at an internal position of the antisensestrand.

In preferred embodiments the iRNA agent includes a first and secondsequences, which are preferably two separate molecules as opposed to twosequences located on the same strand, have sufficient complementarity toeach other to hybridize (and thereby form a duplex region), e.g., underphysiological conditions, e.g., under physiological conditions but notin contact with a helicase or other unwinding enzyme.

It is preferred that the first and second sequences be chosen such thatthe ds iRNA agent includes a single strand or unpaired region at one orboth ends of the molecule. Thus, a ds iRNA agent contains first andsecond sequences, preferable paired to contain an overhang, e.g., one ortwo 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides.Most embodiments will have a 3′ overhang. Preferred sRNA agents willhave single-stranded overhangs, preferably 3′ overhangs, of 1 orpreferably 2 or 3 nucleotides in length at each end. The overhangs canbe the result of one strand being longer than the other, or the resultof two strands of the same length being staggered. 5′ ends arepreferably phosphorylated.

Other modifications to sugars, bases, or backbones described herein canbe incorpoated into the iRNA agents.

The iRNA agents can take an architecture or structure described herein.The iRNA agents can be palindromic, or double targeting, as describedherein.

The iRNA agents can have a sequence such that a non-cannonical or otherthan cannonical Watson-Crick structure is formed between two monomers ofthe iRNA agent or between a strand of the iRNA agent and anothersequence, e.g., a target or off-target sequence, as is described herein.

The iRNA agent can be selected to target any of a broad spectrum ofgenes, including any of the genes described herein.

In a preferred embodiment the iRNA agent has an architecture(architecture refers to one or more of overall length, length of aduplex region, the presence, number, location, or length of overhangs,single strand versus double strand form) described herein. E.g., theiRNA agent can be less than 30 nucleotides in length, e.g., 21-23nucleotides. Preferably, the iRNA is 21 nucleotides in length and thereis a duplex region of about 19 pairs. In one embodiment, the iRNA is 21nucleotides in length, and the duplex region of the iRNA is 19nucleotides. In another embodiment, the iRNA is greater than 30nucleotides in length.

In some embodiment the duplex region of the iRNA agent will have,mismatches. Preferably it will have no more than 1, 2, 3, 4, or 5 bases,which do not form canonical Watson-Crick pairs or which do nothybridize. Overhangs are discussed in detail elsewhere herein but arepreferably about 2 nucleotides in length. The overhangs can becomplementary to the gene sequences being targeted or can be othersequence. TT is a preferred overhang sequence. The first and second iRNAagent sequences can also be joined, e.g., by additional bases to form ahairpin, or by other non-base linkers.

In addition of the RRMS-containing bases the iRNA agents describedherein can include nuclease resistant monomers (NRMs).

In another aspect, the invention features an iRNA agent to which isconjugated a lipophilic moiety, e.g., cholesterol, e.g., by conjugationto an RRMS of an iRNA agent. In a preferred embodiment, the lipophilicmoiety enhances entry of the iRNA agent into a cell. In a preferredembodiment, the cell is part of an organism, tissue, or cell line, e.g.,a primary cell line, immortalized cell line, or any type of cell linedisclosed herein. Thus, the conjugated iRNA agent an be used to silencea target gene in an organism, e.g., a mammal, e.g., a human, or tosilence a target gene in a cell line or in cells which are outside anorganism.

The lipophilic moiety can be chosen, for example, from the groupconsisting of a lipid, cholesterol, oleyl, retinyl, cholesterylresidues, cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. A preferredlipophilic moiety is cholesterol.

The iRNA agent can have a first strand and a second strand, wherein atleast one subunit having formula (I) or formula (II) is incorporatedinto at least one of the strands. The iRNA agent can have one or more ofany of the features described herein. For example, when the subunit isof formula (I), R^(d) can be cholesterol; X can be N(CO)R⁷ or NR⁷, Y canbe CR⁹R¹⁰, and Z can be absent, and R¹ can be (CH₂)_(n)OR^(b) and R³ canbe OR^(b); X can be N(CO)R⁷ or NR⁷, Y can be CR⁹R¹⁰, and Z can beCR¹¹R¹², and R⁹ can be (CH₂)_(n)OR^(b) and R¹⁰ can be OR^(a); X can beN(CO)R⁷ or NR⁷, Y can be NR⁸, and Z can be CR¹¹R¹², and R¹ can be(CH₂)_(n)OR^(b) and R³ can be OR^(a); X can be CH₂; Y can be CR⁹R¹⁰; andZ can be CR¹¹R¹², in which R⁶ can be C(O)NHR⁷; or X can be CH₂; Y can beCR⁹R¹⁰; and Z can be CR¹¹R¹², in which R¹¹ or R¹² can be C(O)NHR⁷ or R⁵and R¹¹ together can be C₅ or C₆ cycloalkyl substituted with N(CH3)R⁷.

In a preferred embodiment, the lipophilic moiety, e.g., a cholesterol,enhances entry of the iRNA agent into a synoviocyte, myocyte,keratinocyte, hepatocyte, leukocyte, endothelial cell (e.g., a kidneycell), B-cell, T-cell, epithelial cell, mesodermal cell, myeloid cell,neural cell, neoplastic cell, mast cell, or fibroblast cell. In certainaspects, a myocyte can be a smooth muscle cell or a cardiac myocyte, afibroblast cell can be a dermal fibroblast, and a leukocyte can be amonocyte. In another preferred embodiment, the cell can be from anadherent tumor cell line derived from a tissue, such as bladder, lung,breast, cervix, colon, pancreas, prostate, kidney, liver, skin, ornervous system (e.g., central nervous system).

In a preferred embodiment, the iRNA agent targets a protein tyrosinephosphatase (PTP-1B) gene or a MAP kinase gene, such as ERK1, ERK2,JNK1, JNK2, or p38. In a preferred embodiment, these iRNA agents areused to silence genes in a fibroblast cell.

In a preferred embodiment, the iRNA agent targets an MDR, Myc, Myb,c-Myc, N-Myc, L-Myc, c-Myb, a-Myb, b-Myb, v-Myb, cyclin D1, Cyclin D2,cyclin E, CDK4, cdc25A, CDK2, or CDK4 gene. In a preferred embodiment,these iRNA agents are used to silence genes in an epithelial cell ormesodermal cell.

In a preferred embodiment, the iRNA agent targets a G72 or DAAO gene. Ina preferred embodiment, these iRNA agents are used to silence genes in aneural cell.

In a preferred embodiment, the iRNA agent targets a gene of thetelomerase pathway, such as a TERT or TR/TERC. In a preferredembodiment, these iRNA agents are used to silence genes in akeratinocyte.

In a preferred embodiment, the iRNA agent targets an interleukin gene,such as IL-1, IL-2, IL-5, IL-8, IL-10, IL-15, IL-16, IL-17, or IL-18. Inanother preferred embodiment, the iRNA agent targets an interleukinreceptor gene, or a chromosomal translocation, such as BCR-ABL,TEL-AML-1, EWS-FLI1, EWS-ERG, TLS-FUS, PAX3-FKHR, or AML-ETO. In apreferred embodiment, these iRNA agents are used to silence genes in alymphoma or a leukemia cell.

In a preferred embodiment, the iRNA agent targets a GRB2 associatedbinding protein. In a preferred embodiment, these iRNA agents are usedto silence genes in a mast cell.

In a preferred embodiment, the iRNA agent targets a growth factor orgrowth factor receptor, such as a TGFbeta or TGFbeta Receptor; PDGF orPDGFR; VEGF or VEGFr1, VEGFr2, or VEGFr3; or IGF-1R, DAF-2, or 1nR. Inanother preferred embodiment, the iRNA agent targets PRL1, PRL2, PRL3,protein kinase C(PKC), PKC receptor, MDR1, TERT, TR/TERC, cyclin D1,NF-KappaB, REL-A, REL-B, PCNA, CHK-1, c-fos, jun, or BCL-2. In apreferred embodiment, these iRNA agents are used to silence genes in anadherent tumor cell line.

In a preferred embodiment, the iRNA agent targets an exogenous gene of agenetically modified cell. An exogenous gene can be, for example, aviral or bacterial gene that derives from an organism that has invadedor infected the cell, or the exogenous gene can be any gene introducedinto the cell by natural or artificial means, such as by a geneticrecombination event. An iRNA agent can target a viral gene, for example,such as a hepatitis viral gene (e.g., a gene of an HAV, HBV, or HCV).Alternatively, or in addition, the iRNA agent can silence a reportergene, such as GFP or beta galatosidase and the like. These iRNA agentscan be used to silence exogenous genes in an adherent tumor cell line.

In a preferred embodiment, the iRNA agent to which the lipophilic moietyis conjugated silences at least one gene, e.g., any gene describedherein, in any one of a number of cell lines including, but not limitedto, a 3T3, DLD2, THP1, Raw264.7, IC₂₁, P388D1, U937, HL60, SEM-K2,WEHI-231, HB56, TIB55, Jurkat, J45.01, K562, EL4, LRMB, Bc1-1, BC-3,TF1, CTLL-2, C1R, Rat6, VERO, MRCS, CV1, Cos 7, RPTE, A10, T24, J82,A549, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, A375,C8161, CCRF-CEM, MCF-7, MDA-MB-231, MOLT, mIMCD-3, NHDF, HeLa, HeLa-S3,Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3,LNCaP, HepG2, or U87 cell line. Cell lines are available from a varietyof sources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassus, Va.)).

In another aspect, the invention provides, methods of silencing a targetgene by providing an iRNA agent to which a lipophilic moiety isconjugated, e.g., a lipophilic conjugated iRNA agent described herein,to a cell. In a preferred embodiment the conjugated iRNA agent an beused to silence a target gene in an organism, e.g., a mammal, e.g., ahuman, or to silence a target gene in a cell line or in cells which areoutside an organism. In the case of a whole organism, the method can beused to silence a gene, e.g., a gene described herein, and treat acondition mediated by the gene. In the case of use on a cell which isnot part of an organism, e.g., a primary cell line, secondary cell line,tumor cell line, or transformed or immortalized cell line, the iRNAagent to which a lipophilic moiety is conjugated can be used to silencea gene, e.g., one described herein. Cells which are not part of a wholeorganism can be used in an initial screen to determine if an iRNA agentis effective in silencing a gene. A test in cells which are not part ofa whole organism can be followed by testing the iRNA agent in a wholeanimal. In preferred embodiments, the iRNA agent which is conjugated toa lipophilic moiety is administered to an organism, or contacted with acell which is not part of an organism, in the absence of (or in areduced amount of) other reagents that facilitate or enhance delivery,e.g., a compound which enhances transit through the cell membrane. (Areduced amount can be an amount of such reagent which is reduced incomparison to what would be needed to get an equal amount ofnonconjugated iRNA agent into the target cell). E.g., the iRNA agentwhich is conjugated to a lipophilic moiety is administered to anorganism, or contacted with a cell which is not part of an organism, inthe absence (or reduced amount) of: an additional lipophilic moiety; atransfection agent, e.g., concentrations of an ion or other substancewhich substantially alters cell permeability to an iRNA agent; atransfecting agent such as Lipofectamine™ (Invitrogen, Carlsbad,Calif.), Lipofectamine 2000™, TransIT-TKO™ (Mirus, Madison, Wis.),FuGENE 6 (Roche, Indianapolis, Ind.), polyethylenimine, X-tremeGENE Q2(Roche, Indianapolis, Ind.), DOTAP, DOSPER, Metafectene™ (Biontex,Munich, Germany), and the like.

In a preferred embodiment the iRNA agent is suitable for delivery to acell in vivo, e.g., to a cell in an organism. In another aspect, theiRNA agent is suitable for delivery to a cell in vitro, e.g., to a cellin a cell line.

An iRNA agent to which a lipophilic moiety is attached can target anygene described herein and can be delivered to any cell type describedherein, e.g., a cell type in an organism, tissue, or cell line. Deliveryof the iRNA agent can be in vivo, e.g., to a cell in an organism, or invitro, e.g., to a cell in a cell line.

In another aspect, the invention provides compositions of iRNA agentsdescribed herein, and in particular compositions of an iRNA agent towhich a lipophilic moiety is conjugated, e.g., a lipophilic conjugatediRNA agent described herein. In a preferred embodiment the compositionis a pharmaceutically acceptable composition.

In preferred embodiments, the composition, e.g., pharmaceuticallyacceptable composition, is free of, has a reduced amount of, or isessentially free of other reagents that facilitate or enhance delivery,e.g., compounds which enhance transit through the cell membrane. (Areduced amount can be an amount of such reagent which is reduced incomparison to what would be needed to get an equal amount ofnonconjugated iRNA agent into the target cell). E.g., the composition isfree of, has a reduced amount of, or is essentially free of: anadditional lipophilic moiety; a transfection agent, e.g., concentrationsof an ion or other substance which substantially alters cellpermeability to an iRNA agent; a transfecting agent such asLipofectamine™ (Invitrogen, Carlsbad, Calif.), Lipofectamine 2000™,TransIT-TKO™ (Mirus, Madison, Wis.), FuGENE 6 (Roche, Indianapolis,Ind.), polyethylenimine, X-tremeGENE Q2 (Roche, Indianapolis, Ind.),DOTAP, DOSPER, Metafectene™ (Biontex, Munich, Germany), and the like.

In a preferred embodiment the composition is suitable for delivery to acell in vivo, e.g., to a cell in an organism. In another aspect, theiRNA agent is suitable for delivery to a cell in vitro, e.g., to a cellin a cell line.

The RRMS-containing iRNA agents can be used in any of the methodsdescribed herein, e.g., to target any of the genes described herein orto treat any of the disorders described herein. They can be incorporatedinto any of the formulations, modes of delivery, delivery modalities,kits or preparations, e.g., pharmaceutical preparations, describedherein. E.g, a kit which inlcudes one or more of the iRNA aentsdescribed herein, a sterile container in which the iRNA agent isdiscolsed, and instructions for use.

The methods and compositions of the invention, e.g., the RRSM-containingiRNA agents described herein, can be used with any of the iRNA agentsdescribed herein. In addition, the methods and compositions of theinvention can be used for the treatment of any disease or disorderdescribed herein, and for the treatment of any subject, e.g., anyanimal, any mammal, such as any human.

The methods and compositions of the invention, e.g., the RRMS-containingiRNA agents described herein, can be used with any dosage and/orformulation described herein, as well as with any route ofadministration described herein.

The non-ribose scaffolds, as well as monomers and dimers of the RRMSsdescribed herein are within the invention

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, ornucleoside surrogate, all of which are defined herein, see the sectionherein entitled RNA Agents. While numerous modified RNAs and nucleosidesurrogates are described herein, preferred examples include those whichinclude one or more RRMS. Preferred examples are those which also a 2′sugar modification, a modification in a single strand overhang,preferably a 3′ single strand overhang, or, particularly if singlestranded, a 5′ modification which includes one or more phosphate groupsor one or more analogs of a phosphate group.

An “iRNA agent” as used herein, is an RNA agent which can, or which canbe cleaved into an RNA agent which can, down regulate the expression ofa target gene, preferably an endogenous or pathogen target RNA. Whilenot wishing to be bound by theory, an iRNA agent may act by one or moreof a number of mechanisms, including post-transcriptional cleavage of atarget mRNA sometimes referred to in the art as RNAi, orpre-transcriptional or pre-translational mechanisms. An iRNA agent caninclude a single strand or can include more than one strands, e.g., itcan be a double stranded iRNA agent. If the iRNA agent is a singlestrand it is particularly preferred that it include a 5′ modificationwhich includes one or more phosphate groups or one or more analogs of aphosphate group.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features andadvantages of the invention will be apparent from the description anddrawings, and from the claims. This application incorporates all citedreferences, patents, and patent applications by references in theirentirety for all purposes.

DESCRIPTION OF DRAWINGS

FIG. 1 a general synthetic scheme for incorporation of RRMS monomersinto an oligonucleotide.

FIG. 2A is a list of substituents that may be present on silicon inOFG¹.

FIG. 2B is a list of substituents that may be present on theC2′-orthoester group.

FIG. 3 is list of representative RRMS cyclic carriers. Panel 1 showspyrroline-based RRMSs; panel 2 shows 3-hydroxyproline-based RRMSs; panel3 shows piperidine-based RRMSs; panel 4 shows morpholine andpiperazine-based RRMSs; and panel 5 shows decalin-based RRMSs. R1 issuccinate or phosphoramidate and R2 is H or a conjugate ligand.

FIG. 4 is a general reaction scheme for 3′ conjugation of peptide intoiRNA.

FIG. 5 is a general reaction scheme for 5′ conjugation of peptide intoiRNA.

FIG. 6 is a general reaction scheme for the synthesis of aza-peptides.

FIG. 7 is a general reaction scheme for the synthesis of N-methyl aminoacids and peptides.

FIG. 8 is a general reaction scheme for the synthesis of 13-methyl aminoacids and Ant and Tat peptides.

FIG. 9 is a general reaction scheme for the synthesis of Ant and Tatoligocarbamates.

FIG. 10 is a general reaction scheme for the synthesis of Ant and Tatoligoureas.

FIG. 11 is a schematic representation of peptide carriers.

FIG. 12 is a structural representation of base pairing inpsuedocomplementary siRNA².

FIG. 13 is a schematic representation of dual targeting siRNAs designedto target the HCV genome.

FIG. 14 is a schematic representation of psuedocomplementary,bifunctional siRNAs designed to target the HCV genome.

FIG. 15 is a list of control and candidate iRNA agents. SEQ ID NOs forthe sense and antisense strands of the duplexes are as follows (sensestrand/antisense strand): Duplex #1 (SEQ ID NO. 29/SEQ ID NO. 30),Duplex #2 (SEQ ID NO. 31/SEQ ID NO. 32), Duplex #3 (SEQ ID NO. 33/SEQ IDNO. 34), Duplex #4 (SEQ ID NO. 35/SEQ ID NO. 36), Duplex #5 (SEQ ID NO.37/SEQ ID NO. 38), Duplex #6 (SEQ ID NO. 55/SEQ ID NO. 56), Duplex #7(SEQ ID NO. 57/SEQ ID NO. 58), Duplex #8 (SEQ ID NO. 59/SEQ ID NO. 60),Duplex #9 (SEQ ID NO. 45/SEQ ID NO. 46), Duplex #10 (SEQ ID NO. 47/SEQID NO. 48), Duplex #11 (SEQ ID NO. 49/SEQ ID NO. 50), and Duplex #12(SEQ ID NO. 51/SEQ ID NO. 52).

FIG. 16 is a graphical representation of relative cell viabilityresults.

FIG. 17 is a graphical representation of gene silencing activityresults.

FIG. 18. is a list of representative cholesterol-tethered RRMS monomers.

FIG. 19 shows LCMS data for a 3′ cholesterol conjugate after PAGEpurification.

FIG. 20 is a graphical representation of Luc silencing with notransfection reagent.

FIG. 21 is a denaturing gel analysis of the human serum stability assayfor AL-DUP-1000. C is the 4 hour time point for siRNA duplex incubatedin PBS buffer alone, OH— is the partial alkaline hydrolysis marker,*s/as represents siRNA duplex containing 5′ end-labeled sense RNA ands/*as represents duplex containing 5′ end-labeled antisense RNA. Sampleswere incubated in 90% human serum and time points were assayed at 10seconds, 5 min, 15 min, 30 min, 1 hour, 2 hours and 4 hours. Black linesto the right of bands indicate exonucleolytic degradation fragments andthe red lines highlight a few of the endonucleolytic degradationfragment.

FIG. 22A is a denaturing gel analysis of the human serum stability assayfor AL-DUP-1393. C is the 4 hour time point for each siRNA duplexincubated in PBS buffer alone, *s/as represents siRNA duplex containing5′ end-labeled sense RNA and s/*as represents duplex containing 5′end-labeled antisense RNA. Samples were assayed at 10 seconds, 15 min,30 min, 1 hour, 2 hours and 4 hours.

FIG. 22B is a denaturing gel analysis of the human serum stability assayfor AL-DUP-1329. The lanes are labeled and the experiment was performedas described for FIG. 22A.

FIG. 23 is a denaturing gel analysis of AL-DUP-1036, AL-DUP-13ff, andAL-DUP-1363 (see Table 8 for sequences). Black vertical lines highlightregions where exonuclease cleavage is suppressed, stars indicate sitesof strong endonucleolytic cleavage in the antisense strand and weakerendonucleolytic cleavage in the sense strand. C is the 4 hour time pointfor each siRNA duplex incubated in PBS buffer alone, *s/as representssiRNA duplex containing 5′ end-labeled sense RNA and s/*as representsduplex containing 5′ end-labeled antisense RNA. Samples were assayed at10 seconds, 15 min, 30 min, 1 hour, 2 hours and 4 hours.

FIG. 24. Human serum stability profile of siRNA duplexes containingcationic modifications. Denaturing gel analysis of AL-DUP-10aa(alkylamino-dT), AL-DUP-lccc (abasic pyrrolidine cationic), andAL-DUP-1403 (see Table 9 for sequences). Black line highlights regionwhere exonuclease cleavage is suppressed and red star indicates site ofstrong endonucleolytic cleavage in the antisense strand. C is the 4 hourtime point for each siRNA duplex incubated in PBS buffer alone, *s/asrepresents siRNA duplex containing 5′ end-labeled sense RNA and s/*asrepresents duplex containing 5′ end-labeled antisense RNA. Samples wereassayed at 10 seconds, 15 min, 30 min, 1 hour, 2 hours and 4 hours.

FIG. 25 is a denaturing gel analysis of the human serum stability assayfor AL-DUP-1069. The black vertical line highlights the region whereexonuclease cleavage is suppressed. C is the 4 hour time point for eachsiRNA duplex incubated in PBS buffer alone, *s/as represents siRNAduplex containing 5′ end-labeled sense RNA and s/*as represents duplexcontaining 5′ end-labeled antisense RNA. Samples were assayed at 10seconds, 15 min, 30 min, 1 hour, 2 hours and 4 hours.

DETAILED DESCRIPTION

Double-stranded (dsRNA) directs the sequence-specific silencing of mRNAthrough a process known as RNA interference (RNAi). The process occursin a wide variety of organisms, including mammals and other vertebrates.

It has been demonstrated that 21-23 nt fragments of dsRNA aresequence-specific mediators of RNA silencing, e.g., by causing RNAdegradation. While not wishing to be bound by theory, it may be that amolecular signal, which may be merely the specific length of thefragments, present in these 21-23 nt fragments recruits cellular factorsthat mediate RNAi. Described herein are methods for preparing andadministering these 21-23 nt fragments, and other iRNAs agents, andtheir use for specifically inactivating gene function. The use of iRNAsagents (or recombinantly produced or chemically synthesizedoligonucleotides of the same or similar nature) enables the targeting ofspecific mRNAs for silencing in mammalian cells. In addition, longerdsRNA agent fragments can also be used, e.g., as described below.

Although, in mammalian cells, long dsRNAs can induce the interferonresponse which is frequently deleterious, sRNAs do not trigger theinterferon response, at least not to an extent that is deleterious tothe cell and host. In particular, the length of the iRNA agent strandsin an sRNA agent can be less than 31, 30, 28, 25, or 23 nt, e.g.,sufficiently short to avoid inducing a deleterious interferon response.Thus, the administration of a composition of sRNA agent (e.g.,formulated as described herein) to a mammalian cell can be used tosilence expression of a target gene while circumventing the interferonresponse. Further, use of a discrete species of iRNA agent can be usedto selectively target one allele of a target gene, e.g., in a subjectheterozygous for the allele.

Moreover, in one embodiment, a mammalian cell is treated with an iRNAagent that disrupts a component of the interferon response, e.g., doublestranded RNA (dsRNA)-activated protein kinase PKR. Such a cell can betreated with a second iRNA agent that includes a sequence complementaryto a target RNA and that has a length that might otherwise trigger theinterferon response.

In a typical embodiment, the subject is a mammal such as a cow, horse,mouse, rat, dog, pig, goat, or a primate. The subject can be a dairymammal (e.g., a cow, or goat) or other farmed animal (e.g., a chicken,turkey, sheep, pig, fish, shrimp). In a much preferred embodiment, thesubject is a human, e.g., a normal individual or an individual that has,is diagnosed with, or is predicted to have a disease or disorder.

Further, because iRNA agent mediated silencing persists for several daysafter administering the iRNA agent composition, in many instances, it ispossible to administer the composition with a frequency of less thanonce per day, or, for some instances, only once for the entiretherapeutic regimen. For example, treatment of some cancer cells may bemediated by a single bolus administration, whereas a chronic viralinfection may require regular administration, e.g., once per week oronce per month.

A number of exemplary routes of delivery are described that can be usedto administer an iRNA agent to a subject. In addition, the iRNA agentcan be formulated according to an exemplary method described herein.

Ligand-Conjugated Monomer Subunits and Monomers for OligonucleotideSynthesis DEFINITIONS

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine.

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁-C₁₂ alkyl indicates that the group may have from1 to 12 (inclusive) carbon atoms in it. The term “haloalkyl” refers toan alkyl in which one or more hydrogen atoms are replaced by halo, andincludes alkyl moieties in which all hydrogens have been replaced byhalo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may beoptionally inserted with O, N, or S. The terms “aralkyl” refers to analkyl moiety in which an alkyl hydrogen atom is replaced by an arylgroup. Aralkyl includes groups in which more than one hydrogen atom hasbeen replaced by an aryl group. Examples of “aralkyl” include benzyl,9-fluorenyl, benzhydryl, and trityl groups.

The term “alkenyl” refers to a straight or branched hydrocarbon chaincontaining 2-8 carbon atoms and characterized in having one or moredouble bonds. Examples of a typical alkenyl include, but not limited to,allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. The term“alkynyl” refers to a straight or branched hydrocarbon chain containing2-8 carbon atoms and characterized in having one or more triple bonds.Some examples of a typical alkynyl are ethynyl, 2-propynyl, and3-methylbutynyl, and propargyl. The sp² and sp³ carbons may optionallyserve as the point of attachment of the alkenyl and alkynyl groups,respectively.

The terms “alkylamino” and “dialkylamino” refer to —NH(alkyl) and —N(alkyl)₂ radicals respectively. The term “aralkylamino” refers to a—NH(aralkyl) radical. The term “alkoxy” refers to an —O-alkyl radical,and the terms “cycloalkoxy” and “aralkoxy” refer to an —O-cycloalkyl andO-aralkyl radicals respectively. The term “siloxy” refers to aR₃SiO-radical. The term “mercapto” refers to an SH radical. The term“thioalkoxy” refers to an —S-alkyl radical.

The term “alkylene” refers to a divalent alkyl (i.e., —R—), e.g., —CH₂—,—CH₂CH₂—, and —CH₂CH₂CH₂—. The term “alkylenedioxo” refers to a divalentspecies of the structure —O—R—O—, in which R represents an alkylene.

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system, wherein any ring atom can be substituted.Examples of aryl moieties include, but are not limited to, phenyl,naphthyl, anthracenyl, and pyrenyl.

The term “cycloalkyl” as employed herein includes saturated cyclic,bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12carbons, wherein any ring atom can be substituted.

The cycloalkyl groups herein described may also contain fused rings.Fused rings are rings that share a common carbon-carbon bond or a commoncarbon atom (e.g., spiro-fused rings). Examples of cycloalkyl moietiesinclude, but are not limited to, cyclohexyl, adamantyl, and norbornyl.

The term “heterocyclyl” refers to a nonaromatic 3-10 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively), whereinany ring atom can be substituted. The heterocyclyl groups hereindescribed may also contain fused rings. Fused rings are rings that sharea common carbon-carbon bond or a common carbon atom (e.g., spiro-fusedrings). Examples of heterocyclyl include, but are not limited totetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino,pyrrolinyl and pyrrolidinyl.

The term “cycloalkenyl” as employed herein includes partiallyunsaturated, nonaromatic, cyclic, bicyclic, tricyclic, or polycyclichydrocarbon groups having 5 to 12 carbons, preferably 5 to 8 carbons,wherein any ring atom can be substituted. The cycloalkenyl groups hereindescribed may also contain fused rings. Fused rings are rings that sharea common carbon-carbon bond or a common carbon atom (e.g., spiro-fusedrings). Examples of cycloalkenyl moieties include, but are not limitedto cyclohexenyl, cyclohexadienyl, or norbornenyl. The term“heterocycloalkenyl” refers to a partially saturated, nonaromatic 5-10membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclicring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively), whereinany ring atom can be substituted. The heterocycloalkenyl groups hereindescribed may also contain fused rings. Fused rings are rings that sharea common carbon-carbon bond or a common carbon atom (e.g., spiro-fusedrings). Examples of heterocycloalkenyl include but are not limited totetrahydropyridyl and dihydropyran.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein any ring atomcan be substituted. The heteroaryl groups herein described may alsocontain fused rings that share a common carbon-carbon bond.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl,cycloalkenyl, aryl, or heteroaryl group at any atom of that group.Suitable substituents include, without limitation, alkyl, alkenyl,alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO₃H, sulfate,phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy,ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl),S(O)_(n)alkyl (where n is O-2), S(O)_(n) aryl (where n is O-2), S(O)_(n)heteroaryl (where n is O-2), S(O). heterocyclyl (where n is O-2), amine(mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and combinationsthereof), ester (alkyl, aralkyl, heteroaralkyl), amide (mono-, di-,alkyl, aralkyl, heteroaralkyl, and combinations thereof), sulfonamide(mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof),unsubstituted aryl, unsubstituted heteroaryl, unsubstitutedheterocyclyl, and unsubstituted cycloalkyl. In one aspect, thesubstituents on a group are independently any one single, or any subsetof the aforementioned substituents.

The terms “adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl” andthe like refer to radicals of adenine, cytosine, guanine, thymine, anduracil.

A “protected” moiety refers to a reactive functional group, e.g., ahydroxyl group or an amino group, or a class of molecules, e.g., sugars,having one or more functional groups, in which the reactivity of thefunctional group is temporarily blocked by the presence of an attachedprotecting group. Protecting groups useful for the monomers and methodsdescribed herein can be found, e.g., in Greene, T. W., Protective Groupsin Organic Synthesis (John Wiley and Sons: New York), 1981, which ishereby incorporated by reference.

General

An RNA agent, e.g., an iRNA agent, containing a preferred, butnonlimiting ligand-conjugated monomer subunit is presented as formula(II) below and in the scheme in FIG. 1. The carrier (also referred to insome embodiments as a “linker”) can be a cyclic or acyclic moiety andincludes two “backbone attachment points” (e.g., hydroxyl groups) and aligand. The ligand can be directly attached (e.g., conjugated) to thecarrier or indirectly attached (e.g., conjugated) to the carrier by anintervening tether (e.g., an acyclic chain of one or more atoms; or anucleobase, e.g., a naturally occurring nucleobase optionally having oneor more chemical modifications, e.g., an unusual base; or a universalbase). The carrier therefore also includes a “ligand or tetheringattachment point” for the ligand and tether/tethered ligand,respectively.

The ligand-conjugated monomer subunit may be the 5′ or 3′ terminalsubunit of the RNA molecule, i.e., one of the two “W” groups may be ahydroxyl group, and the other “W” group may be a chain of two or moreunmodified or modified ribonucleotides. Alternatively, theligand-conjugated monomer subunit may occupy an internal position, andboth “W” groups may be one or more unmodified or modifiedribonucleotides. More than one ligand-conjugated monomer subunit may bepresent in a RNA molecule, e.g., an iRNA agent. Preferred positions forinclusion of a tethered ligand-conjugated monomer subunits, e.g., one inwhich a lipophilic moiety, e.g., cholesterol, is tethered to the carrierare at the 3′ terminus, the 5′ terminus, or an internal position of thesense strand.

The modified RNA molecule of formula (II) can be obtained usingoligonucleotide synthetic methods known in the art. In a preferredembodiment, the modified RNA molecule of formula (II) can be prepared byincorporating one or more of the corresponding monomer compounds (see,e.g., A, B, and C below and in the scheme in FIG. 1) into a growingsense or antisense strand, utilizing, e.g., phosphoramidite orH-phosphonate coupling strategies.

The monomers, e.g., a ligand-conjugated monomer, generally include twodifferently functionalized hydroxyl groups (OFG¹ and OFG²), which arelinked to the carrier molecule (see A below and in FIG. 1), and aligand/tethering attachment point. As used herein, the term“functionalized hydroxyl group” means that the hydroxyl proton has beenreplaced by another substituent. As shown in representative structures Band C below and in FIG. 1, one hydroxyl group (OFG¹) on the carrier isfunctionalized with a protecting group (PG). The other hydroxyl group(OFG²) can be functionalized with either (1) a liquid or solid phasesynthesis support reagent (solid circle) directly or indirectly througha linker, L, as in B, or (2) a phosphorus-containing moiety, e.g., aphosphoramidite as in C. The tethering attachment point may be connectedto a hydrogen atom, a suitable protecting group, a tether, or a tetheredligand at the time that the monomer is incorporated into the growingsense or antisense strand (see variable “R” in A below). Thus, thetethered ligand can be, but need not be attached to the monomer at thetime that the monomer is incorporated into the growing strand. Incertain embodiments, the tether, the ligand or the tethered ligand maybe linked to a “precursor” ligand-conjugated monomer subunit after a“precursor” ligand-conjugated monomer subunit has been incorporated intothe strand. The wavy line used below (and elsewhere herein) refers to aconnection, and can represent a direct bond between the moiety and theattachment point or a tethering molecule which is interposed between themoiety and the attachment point. Directly tethered means the moiety isbound directly to the attachment point. Indirectly tethered means thatthere is a tether molecule interposed between the attachment point andthe moiety.

The (OFG¹) protecting group may be selected as desired, e.g., from T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d.Ed., John Wiley and Sons (1991). The protecting group is preferablystable under amidite synthesis conditions, storage conditions, andoligonucleotide synthesis conditions. Hydroxyl groups, —OH, arenucleophilic groups (i.e., Lewis bases), which react through the oxygenwith electrophiles (i.e., Lewis acids). Hydroxyl groups in which thehydrogen has been replaced with a protecting group, e.g., atriarylmethyl group or a trialkylsilyl group, are essentially unreactiveas nucleophiles in displacement reactions. Thus, the protected hydroxylgroup is useful in preventing e.g., homocoupling of compoundsexemplified by structure C during oligonucleotide synthesis. In someembodiments, a preferred protecting group is the dimethoxytrityl group.In other embodiments, a preferred protecting group is a silicon-basedprotecting group having the formula below:

X5′, X5″, and X5′″ can be selected from substituted or unsubstitutedalkyl, cycloalkyl, aryl, araklyl, heteroaryl, alkoxy, cycloalkoxy,aralkoxy, aryloxy, heteroaryloxy, or siloxy (i.e., R₃SiO-, the three “R”groups can be any combination of the above listed groups). X⁵′, X⁵″, andX⁵″ may all be the same or different; also contemplated is a combinationin which two of X⁵′, X⁵″, and X⁵″ are identical and the third isdifferent. In certain embodiments X⁵′, X⁵″, and X⁵″ include at least onealkoxy or siloxy groups and may be any one of the groups listed in FIG.2A, a preferred combination includes X⁵′, X⁵″=trimethylsiloxy andX⁵″=1,3-(triphenylmethoxy)-2-propoxy or cyclododecyloxy.

Other preferred combinations of X⁵′, X⁵″, and X⁵″ include those thatresult in OFG¹ groups that meet the deprotection and stability criteriadelineated below. The group is preferably stable under amidite synthesisconditions, storage conditions, and oligonucleotide synthesisconditions. Rapid removal, i.e., less than one minute, of the silylgroup from e.g., a support-bound oligonucleotide is desirable because itcan reduce synthesis times and thereby reduce exposure time of thegrowing oligonucleotide chain to the reagents. Oligonucleotide synthesiscan be improved if the silyl protecting group is visible duringdeprotection, e.g., from the addition of a chromophore silylsubstituent.

Selection of silyl protecting groups can be complicated by the competingdemands of the essential characteristics of stability and facileremoval, and the need to balance these competitive goals. Mostsubstituents that increase stability can also increase the reaction timerequired for removal of the silyl group, potentially increasing thelevel of difficulty in removal of the group.

The addition of alkoxy and siloxy substituents to OFG¹silicon-containing protecting groups increases the susceptibility of theprotecting groups to fluoride cleavage of the silylether bonds.Increasing the steric bulk of the substituents preserves stability whilenot decreasing fluoride lability to an equal extent. An appropriatebalance of substituents on the silyl group makes a silyl ether a viablenucleoside protecting group.

Candidate OFG¹ silicon-containing protecting groups may be tested byexposing a tetrahydrofuran solution of a preferred carrier bearing thecandidate OFG¹ group to five molar equivalents of tetrahydrofuran atroom temperature. The reaction time may be determined by monitoring thedisappearance of the starting material by thin layer chromatography.

When the OFG² in B includes a linker, e.g., a relatively long organiclinker, connected to a soluble or insoluble support reagent, solution orsolid phase synthesis techniques can be employed to build up a chain ofnatural and/or modified ribonucleotides once OFG¹ is deprotected andfree to react as a nucleophile with another nucleoside or monomercontaining an electrophilic group (e.g., an amidite group).Alternatively, a natural or modified ribonucleotide oroligoribonucleotide chain can be coupled to monomer C via an amiditegroup or H-phosphonate group at OFG². Subsequent to this operation, OFG¹can be deblocked, and the restored nucleophilic hydroxyl group can reactwith another nucleoside or monomer containing an electrophilic group. R′can be substituted or unsubstituted alkyl or alkenyl. In preferredembodiments, R′ is methyl, allyl or 2-cyanoethyl. R¹¹ may a C₁-C₁₀ alkylgroup, preferably it is a branched group containing three or morecarbons, e.g., isopropyl.

OFG² in B can be hydroxyl functionalized with a linker, which in turncontains a liquid or solid phase synthesis support reagent at the otherlinker terminus. The support reagent can be any support medium that cansupport the monomers described herein. The monomer can be attached to aninsoluble support via a linker, L, which allows the monomer (and thegrowing chain) to be solubilized in the solvent in which the support isplaced. The solubilized, yet immobilized, monomer can react withreagents in the surrounding solvent; unreacted reagents and solubleby-products can be readily washed away from the solid support to whichthe monomer or monomer-derived products is attached. Alternatively, themonomer can be attached to a soluble support moiety, e.g., polyethyleneglycol (PEG) and liquid phase synthesis techniques can be used to buildup the chain. Linker and support medium selection is within skill of theart. Generally the linker may be —C(O)(CH₂)_(q)C(O)—, or—C(O)(CH₂)_(q)S—, in which q can be 0, 1, 2, 3, or 4; preferably, it isoxalyl, succinyl or thioglycolyl. Standard control pore glass solidphase synthesis supports can not be used in conjunction with fluoridelabile 5′ silyl protecting groups because the glass is degraded byfluoride with a significant reduction in the amount of full-lengthproduct. Fluoride-stable polystyrene based supports or PEG arepreferred.

The ligand/tethering attachment point can be any divalent, trivalent,tetravalent, pentavalent or hexavalent atom. In some embodiments,ligand/tethering attachment point can be a carbon, oxygen, nitrogen orsulfur atom. For example, a ligand/tethering attachment point precursorfunctional group can have a nucleophilic heteroatom, e.g., —SH, —NH₂,secondary amino, ONH₂, or NH₂NH₂. As another example, theligand/tethering attachment point precursor functional group can be anolefin, e.g., —CH═CH₂, and the precursor functional group can beattached to a ligand, a tether, or tethered ligand using, e.g.,transition metal catalyzed carbon-carbon (for example olefin metathesis)processes or cycloadditions (e.g., Diels-Alder). As a further example,the ligand/tethering attachment point precursor functional group can bean electrophilic moiety, e.g., an aldehyde. When the carrier is a cycliccarrier, the ligand/tethering attachment point can be an endocyclic atom(i.e., a constituent atom in the cyclic moiety, e.g., a nitrogen atom)or an exocyclic atom (i.e., an atom or group of atoms attached to aconstituent atom in the cyclic moiety).

The carrier can be any organic molecule containing attachment points forOFG¹, OFG², and the ligand. In certain embodiments, carrier is a cyclicmolecule and may contain heteroatoms (e.g., O, N or S). E.g., carriermolecules may include aryl (e.g., benzene, biphenyl, etc.), cycloalkyl(e.g., cyclohexane, cis or trans decalin, etc.), or heterocyclyl(piperazine, pyrrolidine, etc.). In other embodiments, the carrier canbe an acyclic moiety, e.g., based on serinol. Any of the above cyclicsystems may include substituents in addition to OFG¹, OFG², and theligand.

Sugar-Based Monomers

In some embodiments, the carrier molecule is an oxygen containingheterocycle. Preferably the carrier is a ribose sugar as shown instructure LCM-I. In this embodiment, the monomer, e.g., aligand-conjugated monomer is a nucleoside.

“B” represents a nucleobase, e.g., a naturally occurring nucleobaseoptionally having one or more chemical modifications, e.g., and unusualbase; or a universal base.

As used herein, an “unusual” nucleobase can include any one of thefollowing:

-   2-methyladeninyl,-   N6-methyladeninyl,-   2-methylthio-N-6-methyladeninyl,-   N6-isopentenyladeninyl,-   2-methylthio-N-6-isopentenyladeninyl,-   N6-(cis-hydroxyisopentenyl)adeninyl,-   2-methylthio-N-6-(cis-hydroxyisopentenyl) adeninyl,-   N6-glycinylcarbamoyladeninyl,-   N6-threonylcarbamoyladeninyl,-   2-methylthio-N-6-threonyl carbamoyladeninyl,-   N6-methyl-N-6-threonylcarbamoyladeninyl,-   N6-hydroxynorvalylcarbamoyladeninyl,-   2-methylthio-N-6-hydroxynorvalyl carbamoyladeninyl,-   N6,N6-dimethyladeninyl,-   3-methylcytosinyl,-   5-methylcytosinyl,-   2-thiocytosinyl,-   5-formylcytosinyl,

-   N4-methylcytosinyl,-   5-hydroxymethylcytosinyl,-   1-methylguaninyl,-   N2-methylguaninyl,-   7-methylguaninyl,-   N2,N2-dimethylguaninyl,

-   N2,7-dimethylguaninyl,-   N2,N2,7-trimethylguaninyl,-   1-methylguaninyl,-   7-cyano-7-deazaguaninyl,-   7-aminomethyl-7-deazaguaninyl,-   pseudouracilyl,-   dihydrouracilyl,-   5-methyluracilyl,-   1-methylpseudouracilyl,-   2-thiouracilyl,-   4-thiouracilyl,-   2-thiothyminyl-   5-methyl-2-thiouracilyl,-   3-(3-amino-3-carboxypropyl)uracilyl,-   5-hydroxyuracilyl,-   5-methoxyuracilyl,-   uracilyl 5-oxyacetic acid,-   uracilyl 5-oxyacetic acid methyl ester,-   5-(carboxyhydroxymethyl)uracilyl,-   5-(carboxyhydroxymethyl)uracilyl methyl ester,-   5-methoxycarbonylmethyluracilyl,-   5-methoxycarbonylmethyl-2-thiouracilyl,-   5-aminomethyl-2-thiouracilyl,-   5-methylaminomethyluracilyl,-   5-methylaminomethyl-2-thiouracilyl,-   5-methylaminomethyl-2-selenouracilyl,-   5-carbamoylmethyluracilyl,-   5-carboxymethylaminomethyluracilyl,-   5-carboxymethylaminomethyl-2-thiouracilyl,-   3-methyluracilyl,-   1-methyl-3-(3-amino-3-carboxypropyl) pseudouracilyl,-   5-carboxymethyluracilyl,-   5-methyldihydrouracilyl, or-   3-methylpseudouracilyl.

A universal base can form base pairs with each of the natural DNA/RNAbases, exhibiting relatively little discrimination between them. Ingeneral, the universal bases are non-hydrogen bonding, hydrophobic,aromatic moieties which can stabilize e.g., duplex RNA or RNA-likemolecules, via stacking interactions. A universal base can also includehydrogen bonding substituents. As used herein, a “universal base” caninclude anthracenes, pyrenes or any one of the following:

In some embodiments, B can form part of a tether that connects a ligandto the carrier. For example, the tether can beB—CH═CH—C(O)NH—(CH₂)₅—NHC(O)-LIGAND. In a preferred embodiment, thedouble bond is trans, and the ligand is a substituted or unsubstitutedcholesterolyl radical (e.g., attached through the D-ring side chain orthe C-3 hydroxyl); an aralkyl moiety having at least one sterogeniccenter and at least one substituent on the aryl portion of the aralkylgroup; or a nucleobase. In certain embodiments, B, in the tetherdescribed above, is uracilyl or a universal base, e.g., an aryl moiety,e.g., phenyl, optionally having additional substituents, e.g., one ormore fluoro groups. B can be substituted at any atom with the remainderof the tether.

X² can include “oxy” or “deoxy” substituents in place of the 2′ —OH; orbe a ligand or a tethered ligand.

Examples of “oxy”-substituents include alkoxy or aryloxy (OR, e.g., R═H,alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, sugar, or protectinggroup); polyethyleneglycols (PEG), O(CH₂CH₂O).CH₂CH₂OR; “locked” nucleicacids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylenebridge, to the 4′ carbon of the same ribose sugar; O—PROTECTED AMINE(AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)_(n)PROTECTED AMINE, (e.g., AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino),and orthoester. Amine protecting groups can include formyl, amido,benzyl, allyl, etc.

Preferred orthoesters have the general formula J. The groups R³¹ and R³²may be the same or different and can be any combination of the groupslisted in FIG. 2B. A preferred orthoester is the “ACE” group, shownbelow as structure K.

“Deoxy” substituents include hydrogen (i.e. deoxyribose sugars); halo(e.g., fluoro); protected amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid in which all amino are protected); fully protectedpolyamino (e.g., NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE, wherein AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino and all amino groups areprotected), —NHC(O)R(R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar), cyano; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl,aryl, alkenyl and alkynyl, which may be optionally substituted withe.g., a protected amino functionality. Preferred substitutents are2′-methoxyethyl, 2′-OCH₃,2′-O-allyl, 2′-C— allyl, and 2′-fluoro.

X³ is as described for OFG² above. PG can be a triarylmethyl group(e.g., a dimethoxytrityl group) or Si(X⁵′)(X⁵″)(X⁵′″) in which(X⁵′),(X⁵″), and (X⁵′″) are as described elsewhere.

Sugar Replacement-Based Monomers, e.g., Ligand-Conjugated Monomers(Cyclic)

Cyclic sugar replacement-based monomers, e.g., sugar replacement-basedligand-conjugated monomers, are also referred to herein as ribosereplacement monomer subunit (RRMS) monomer compounds. Preferred carriershave the general formula (LCM-2) provided below (In that structurepreferred backbone attachment points can be chosen from R¹ or R²; R³ orR⁴; or R⁹ and R¹⁰ if Y is CR⁹R¹⁰ (two positions are chosen to give twobackbone attachment points, e.g., R¹ and R⁴, or R⁴ and R⁹)). Preferredtethering attachment points include R⁷; R⁵ or R⁶ when X is CH₂. Thecarriers are described below as an entity, which can be incorporatedinto a strand. Thus, it is understood that the structures also encompassthe situations wherein one (in the case of a terminal position) or two(in the case of an internal position) of the attachment points, e.g., R¹or R²; R³ or R⁴; or R⁹ or R¹⁰ (when Yis)CR⁹R¹⁰, is connected to thephosphate, or modified phosphate, e.g., sulfur containing, backbone.E.g., one of the above-named R groups can be —CH₂—, wherein one bond isconnected to the carrier and one to a backbone atom, e.g., a linkingoxygen or a central phosphorus atom.)

in which,

X is N(CO)R⁷, NR⁷ or CH₂;

Y is NR⁸, O, S, CR⁹R¹⁰;

Z is CR¹¹R¹² or absent;

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^(a), or(CH₂)_(n)OR^(b), provided that at least two of R¹, R², R³, R⁴, R⁹, andR¹⁰ are OR^(a) and/or (CH₂)_(n)OR^(b);

Each of R⁵, R⁶, R¹¹ , and R¹² is, independently, a ligand, H, C₁-C₆alkyl optionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹together are C₃-C₈ cycloalkyl optionally substituted with R¹⁴;

R⁷ can be a ligand, e.g., R⁷ can be R^(d), or R⁷ can be a ligandtethered indirectly to the carrier, e.g., through a tethering moiety,e.g., C₁-C₂₀ alkyl substituted with NR^(c)R^(d); or C₁-C₂₀ alkylsubstituted with NHC(O)R^(d);

R⁸ is H or C₁-C₆ alkyl;

R¹³ is hydroxy, C₁-C₄ alkoxy, or halo;

R¹⁴ is NR⁶R⁷;

R¹⁵ is C₁-C₆ alkyl optionally substituted with cyano, or C₂-C₆ alkenyl;

R¹⁶ is C₁-C₁₀ alkyl;

R¹⁷ is a liquid or solid phase support reagent;

L is —C(O)(CH₂)_(q)C(O)—, or —C(O)(CH₂)_(q)S—;

R^(a) is a protecting group, e.g., CAr₃; (e.g., a dimethoxytrityl group)or Si(X⁵′)(X⁵″)(X⁵′″) in which (X⁵′),(X⁵″), and (X⁵′″) are as describedelsewhere.

R^(b) is P(O)(O⁻)H, P(OR¹⁵)N(R¹⁰)₂ or L-R¹⁷;

R⁶ is H or C₁-C₆ alkyl;

R^(d) is H or a ligand;

Each Ar is, independently, C₆-C₁₀ aryl optionally substituted with C₁-C₄alkoxy;

n is 1-4; and q is O-4.

Exemplary carriers include those in which, e.g., X is N(CO)R⁷ or NR⁷, Yis CR⁹R¹⁰, and Z is absent; or X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Zis CR¹¹R¹²; or X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹²; or X isN(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹²; or X is CH₂; Y is CR⁹R¹⁰; Z isCR¹¹R¹², R⁵ and R¹¹ together form C₆ cycloalkyl (H, z=2), or the indanering system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹²; and R⁵ and R¹¹together form C₅ cycloalkyl (H, z=1).

In certain embodiments, the carrier may be based on the pyrroline ringsystem or the 4-hydroxyproline ring system, e.g., X is N(CO)R⁷ or NR⁷, Yis CR⁹R¹⁰, and Z is absent (D). OFG¹ is preferably attached to a primarycarbon, e.g., an exocyclic alkylene

group, e.g., a methylene group, connected to one of the carbons in thefive-membered ring (—CH₂OFG¹ in D). OFG² is preferably attached directlyto one of the carbons in the five-membered ring (—OFG² in D). For thepyrroline-based carriers, —CH₂OFG¹ may be attached to C-2 and OFG² maybe attached to C-3; or —CH₂OFG¹ may be attached to C-3 and OFG² may beattached to C-4. In certain embodiments, CH₂OFG¹ and OFG² may begeminally substituted to one of the above-referenced carbons. For the3-hydroxyproline-based carriers, —CH₂OFG¹ may be attached to C-2 andOFG² may be attached to C-4. The pyrroline- and 4-hydroxyproline-basedmonomers may therefore contain linkages (e.g., carbon-carbon bonds)wherein bond rotation is restricted about that particular linkage, e.g.restriction resulting from the presence of a ring. Thus, CH₂OFG¹ andOFG² may be cis or trans with respect to one another in any of thepairings delineated above. Accordingly, all cis/trans isomers areexpressly included. The monomers may also contain one or more asymmetriccenters and thus occur as racemates and racemic mixtures, singleenantiomers, individual diastereomers and diastereomeric mixtures. Allsuch isomeric forms of the monomers are expressly included (e.g., thecenters bearing CH₂OFG¹ and OFG² can both have the R configuration; orboth have the S configuration; or one center can have the Rconfiguration and the other center can have the S configuration and viceversa). The tethering attachment point is preferably nitrogen. Preferredexamples of carrier D include the following:

In certain embodiments, the carrier may be based on the piperidine ringsystem (E), e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹².OFG¹ is preferably

attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., amethylene group (n=1) or ethylene group (n=2), connected to one of thecarbons in the six-membered ring [—(CH₂)_(n)OFG¹ in E]. OFG² ispreferably attached directly to one of the carbons in the six-memberedring (—OFG² in E).—(CH₂)_(n)OFG¹ and OFG² may be disposed in a geminalmanner on the ring, i.e., both groups may be attached to the samecarbon, e.g., at C-2, C-3, or C-4. Alternatively, —(CH₂)_(n)OFG¹ andOFG² may be disposed in a vicinal manner on the ring, i.e., both groupsmay be attached to adjacent ring carbon atoms, e.g.,

—(CH₂)_(n)OFG¹ may be attached to C-2 and OFG² may be attached to C-3;—(CH₂)_(n)OFG¹ may be attached to C-3 and OFG² may be attached to C-2;—(CH₂)_(n)OFG¹ may be attached to C-3 and OFG² may be attached to C-4;or —(CH₂)_(n)OFG¹ may be attached to C-4 and OFG² may be attached toC-3. The piperidine-based monomers may therefore contain linkages (e.g.,carbon-carbon bonds) wherein bond rotation is restricted about thatparticular linkage, e.g. restriction resulting from the presence of aring. Thus, —(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect toone another in any of the pairings delineated above. Accordingly, allcis/trans isomers are expressly included. The monomers may also containone or more asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of the monomers areexpressly included (e.g., the centers bearing CH₂OFG¹ and OFG² can bothhave the R configuration; or both have the S configuration; or onecenter can have the R configuration and the other center can have the Sconfiguration and vice versa). The tethering attachment point ispreferably nitrogen.

In certain embodiments, the carrier may be based on the piperazine ringsystem (F), e.g., X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹², orthe morpholine ring system (G), e.g., X is N(CO)R⁷ or NR⁷, Y is O, and Zis CR¹¹R¹². OFG¹ is preferably

attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., amethylene group, connected to one of the carbons in the six-memberedring (—CH₂OFG¹ in F or G). OFG² is preferably attached directly to oneof the carbons in the six-membered rings (—OFG² in F or G). For both Fand G, —CH₂OFG¹ may be attached to C-2 and OFG² may be attached to C-3;or vice versa. In certain embodiments, CH₂OFG¹ and OFG² may be geminallysubstituted to one of the above-referenced carbons. The piperazine- andmorpholine-based monomers may therefore contain linkages (e.g.,carbon-carbon bonds) wherein bond rotation is restricted about thatparticular linkage, e.g. restriction resulting from the presence of aring. Thus, CH₂OFG¹ and OFG² may be cis or trans with respect to oneanother in any of the pairings delineated above. Accordingly, allcis/trans isomers are expressly included. The monomers may also containone or more asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of the monomers areexpressly included (e.g., the centers bearing CH₂OFG¹ and OFG² can bothhave the R configuration; or both have the S configuration; or onecenter can have the R configuration and the other center can have the Sconfiguration and vice versa). R′″ can be, e.g., C₁-C₆ alkyl, preferablyCH₃. The tethering attachment point is preferably nitrogen in both F andG.

In certain embodiments, the carrier may be based on the decalin ringsystem, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹together form C₆ cycloalkyl (H, z=2), or the indane ring system, e.g., Xis CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹ together form C₅cycloalkyl (H, z=1). OFG¹ is preferably attached to a primary carbon,

e.g., an exocyclic methylene group (n=1) or ethylene group (n=2)connected to one of C-2, C-3, C-4, or C-5 [-(CH₂)_(n)OFG¹ in H]. OFG² ispreferably attached directly to one of C-2, C-3, C-4, or C-5 (—OFG² inH).—(CH₂)_(n)OFG¹ and OFG² may be disposed in a geminal manner on thering, i.e., both groups may be attached to the same carbon, e.g., atC-2, C-3, C-4, or C-5. Alternatively, —(CH₂)_(n)OFG¹ and OFG² may bedisposed in a vicinal manner on the ring, i.e., both groups may beattached to adjacent ring carbon atoms, e.g., —(CH₂)_(n)OFG¹ may beattached to C-2 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ may beattached to C-3 and OFG² may be attached to C-2; —(CH₂)_(n)OFG¹ may beattached to C-3 and OFG² may be attached to C-4; or —(CH₂)_(n)OFG¹ maybe attached to C-4 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ maybe attached to C-4 and OFG² may be attached to C-5; or —(CH₂)_(n)OFG¹may be attached to C-5 and OFG² may be attached to C-4. The decalin orindane-based monomers may therefore contain linkages (e.g.,carbon-carbon bonds) wherein bond rotation is restricted about thatparticular linkage, e.g. restriction resulting from the presence of aring. Thus, —(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect toone another in any of the pairings delineated above. Accordingly, allcis/trans isomers are expressly included. The monomers may also containone or more asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of the monomers areexpressly included (e.g., the centers bearing CH₂OFG¹ and OFG² can bothhave the R configuration; or both have the S configuration; or onecenter can have the R configuration and the other center can have the Sconfiguration and vice versa). In a preferred embodiment, thesubstituents at C-1 and C-6 are trans with respect to one another. Thetethering attachment point is preferably C-6 or C-7.

Other carriers may include those based on 3-hydroxyproline (J). Thus,—(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect to one another.Accordingly, all cis/trans isomers are expressly included. The monomersmay also contain one or more asymmetric centers

and thus occur as racemates and racemic mixtures, single enantiomers,individual diastereomers and diastereomeric mixtures. All such isomericforms of the monomers are expressly included (e.g., the centers bearingCH₂OFG¹ and OFG² can both have the R configuration; or both have the Sconfiguration; or one center can have the R configuration and the othercenter can have the S configuration and vice versa). The tetheringattachment point is preferably nitrogen.

Representative cyclic, sugar replacement-based carriers are shown inFIG. 3.

Sugar Replacement-Based Monomers (Acyclic)

Acyclic sugar replacement-based monomers, e.g., sugar replacement-basedligand-conjugated monomers, are also referred to herein as ribosereplacement monomer subunit (RRMS) monomer compounds. Preferred acycliccarriers can have formula LCM-3 or LCM-4 below.

In some embodiments, each of x, y, and z can be, independently of oneanother, 0, 1, 2, or 3. In formula LCM-3, when y and z are different,then the tertiary carbon can have either the R or S configuration. Inpreferred embodiments, x is zero and y and z are each 1 in formula LCM-3(e.g., based on serinol), and y and z are each 1 in formula LCM-3. Eachof formula LCM-3 or LCM-4 below can optionally be substituted, e.g.,with hydroxy, alkoxy, perhaloalkyl.

Tethers

In certain embodiments, a moiety, e.g., a ligand may be connectedindirectly to the carrier via the intermediacy of an intervening tether.Tethers are connected to the carrier at a tethering attachment point(TAP) and may include any C₁-C₁₀₀ carbon-containing moiety, (e.g.C₁-C₇₅, C₁-C₅₀, C₁-C₂₀, C₁-C₁₀; C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, orC₁₀), preferably having at least one nitrogen atom. In preferredembodiments, the nitrogen atom forms part of a terminal amino or amido(NHC(O)—) group on the tether, which may serve as a connection point forthe ligand. Preferred tethers (underlined) include TAP-(CH₂)_(n)NH—;TAP-C(O)(CH₂)_(n)NH—; TAP-NR″″(CH₂)_(n)NH—, TAP-C(O)—(CH₂)_(n)—CO—;TAP-C(O)—(CH₂)_(n)—C(O)O—; TAP-C(O)—O—; TAP-C(O)—(CH₂)_(n)—NH—C(O)—;TAP-C(O)—(CH₂)_(n) ; TAP-C(O)—NH—; TAP-C(O)—; TAP-(CH₂)_(n)—C(O)—;TAP-(CH₂)_(n)C(O)O—; TAP-(CH₂)_(n)—; or TAP-(CH₂)_(n)—NH—C(O)—; in whichn is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20) and R″″ is C₁-C₆ alkyl. Preferably, n is 5, 6, or 11.In other embodiments, the nitrogen may form part of a terminal oxyaminogroup, e.g., —ONH₂, or hydrazino group, —NHNH₂. The tether mayoptionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl,and/or optionally inserted with one or more additional heteroatoms,e.g., N, O, or S. Preferred tethered ligands may include, e.g.,TAP-(CH₂)_(n)NH(LIGAND); TAP-C(O)(CH₂)_(n)NH(LIGAND);TAP-NR″″(CH₂)_(n)NH(LIGAND); TAP-(CH₂)_(n)ON(LIGAND);TAP-C(O)(CH₂)_(n)(LIGAND); TAP-NR″″(CH₂)ONH(LIGAND);TAP-(CH₂)_(n)NHNH₂(LIGAND), TAP-C(O)(CH₂)_(n)NHNH₂(LIGAND);TAP-NR″″(CH₂)_(n)NHNH₂(LIGAND); TAP-(CO)—(CH₂)_(n)—C(O)(LIGAND);TAP-C(O)—(CH₂)_(n)—C(O)O(LIGAND); TAP-C(O)—O(LIGAND);TAP-C(O)—(CH₂)_(n)—NH—C(O)(LIGAND); TAP-C(O)—(CH₂)_(n)(LIGAND);TAP-C(O)—NH(LIGAND); TAP-C(O)(LIGAND); TAP-(CH₂)_(n)C(O)(LIGAND);TAP-(CH₂LIGAND); TAP—(CH₂)_(n)(LIGAND); or TAP-(CH₂)_(n)NH—C(O)(LIAND).In some embodiments, amino terminated tethers (e.g., NH₂, ONH₂, NH₂NH₂)can form an imino bond (i.e., C≡N) with the ligand. In some embodiments,amino terminated tethers (e.g., NH₂, ONH₂, NH₂NH₂) can acylated, e.g.,with C(O)CF₃.

In some embodiments, the tether can terminate with a mercapto group(i.e., SH) or an olefin (e.g., CH═CH₂)_(n) For example, the tether canbe TAP-(CH₂)_(n)—SH, TAP-C(O)(CH₂)_(n)SH, TAP-(CH₂)_(n)—(CH═CH), orTAP-C(O)(CH₂)_(n)(CH═CH₂), in which n can be as described elsewhere. Thetether may optionally be substituted, e.g., with hydroxy, alkoxy,perhaloalkyl, and/or optionally inserted with one or more additionalheteroatoms, e.g., N, O, or S. The double bond can be cis or trans or Eor Z.

In other embodiments the tether may include an electrophilic moiety,preferably at the terminal position of the tether. Preferredelectrophilic moieties include, e.g., an aldehyde, alkyl halide,mesylate, tosylate, nosylate, or brosylate, or an activated carboxylicacid ester, e.g. an NHS ester, or a pentafluorophenyl ester. Preferredtethers (underlined) include TAP—(CH₂)_(n)CHO; TAP-C(O)(CH₂)_(n)CHO; orTAP-NR″″(CH₂)_(n)CHO, in which n is 1-6 and R″″ is C₁-C₆ alkyl; orTAP-(CH₂)_(n)C(O)ONHS; TAP-C(O)(CH₂)_(n)C(O)ONHS; orTAP-NR″″(CH₂)_(n)C(O)ONHS, in which n is 1-6 and R″″ is C₁-C₆ alkyl;TAP-(CH₂)_(n)C(O)OC₆F₅ ; TAP-C(O)(CH₂)_(n)C(O)OC₆F₅ ; orTAP-NR″″(CH₂)_(n)C(O)OC₆F₅ , in which n is 1-11 and R″″ is C₁-C₆ alkyl;or —(CH₂)_(n)CH₂LG; TAP-C(O)(CH₂)_(n)CH₂LG; or TAP-NR″″(CH₂)_(n)LG, inwhich n can be as described elsewhere and R″″ is C₁-C₆ alkyl (LG can bea leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate).Tethering can be carried out by coupling a nucleophilic group of aligand, e.g., a thiol or amino group with an electrophilic group on thetether.

In other embodiments, it can be desirable for the monomer to include aphthalimido group (K) at the terminal position of the tether.

In other embodiments, other protected amino groups can be at theterminal position of the tether, e.g., alloc, monomethoxy trityl (MMT),trifluoroacetyl, Fmoc, or aryl sulfonyl (e.g., the aryl portion can beortho-nitrophenyl or ortho, para-dinitrophenyl).

Any of the tethers described herein may further include one or moreadditional linking groups, e.g., —O—(CH₂)_(n)—, —(CH₂)_(n)—SS—,—(CH₂)_(n)—, or —(CH═CH)—.

Tethered Ligands

A wide variety of entities, e.g., ligands, can be tethered to an iRNAagent, e.g., to the carrier of a ligand-conjugated monomer subunit.Examples are described below in the context of a ligand-conjugatedmonomer subunit but that is only preferred, entities can be coupled atother points to an iRNA agent.

Preferred moieties are ligands, which are coupled, preferablycovalently, either directly or indirectly via an intervening tether, tothe carrier. In preferred embodiments, the ligand is attached to thecarrier via an intervening tether. As discussed above, the ligand ortethered ligand may be present on the ligand-conjugated monomer \ whenthe ligand-conjugated monomer is incorporated into the growing strand.In some embodiments, the ligand may be incorporated into a “precursor”ligand-conjugated monomer subunit after a “precursor” ligand-conjugatedmonomer subunit has been incorporated into the growing strand. Forexample, a monomer having, e.g., an amino-terminated tether, e.g.,TAP-(CH₂)_(n)NH₂ may be incorporated into a growing sense or antisensestrand. In a subsequent operation, i.e., after incorporation of theprecursor monomer subunit into the strand, a ligand having anelectrophilic group, e.g., a pentafluorophenyl ester or aldehyde group,can subsequently be attached to the precursor ligand-conjugated monomerby coupling the electrophilic group of the ligand with the terminalnucleophilic group of the precursor ligand-conjugated monomer subunittether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g, molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; nuclease-resistanceconferring moieties; and natural or unusual nucleobases. Generalexamples include lipophiles, lipids, steroids (e.g., uvaol, hecigenin,diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin,Friedelin, epifriedelanol derivatized lithocholic acid), vitamins (e.g.,folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins,protein binding agents, integrin targeting molecules,polycationics,peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, (e.g., human serumalbumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate(e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin orhyaluronic acid); amino acid, or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid. Examples of polyamino acids include polyaminoacid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationicmoieties, e.g., cationic lipid, cationic porphyrin, quaternary salt of apolyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide orRGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g, cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone, glycerol (e.g., estersand ethers thereof, e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈,C₁₉, or C₂₀ alkyl; e.g., 1,3-bis-O(hexadecyl)glycerol,1,3-bis-O(octaadecyl)glycerol), geranyloxyhexyl group,hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group,palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,bisimidazole, histamine, imidazole clusters, acridine-imidazoleconjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP,or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. Theligand can be, for example, a lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF—X¹³.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the iRNA agent into the cell byactivating an inflammatory response, for example. Exemplary ligands thatwould have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver.

Other molecules that can bind HSA can also be used as ligands. Forexample, neproxin or aspirin can be used. A lipid or lipid-based ligandcan (a) increase resistance to degradation of the conjugate, (b)increase targeting or transport into a target cell or cell membrane,and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alphα-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and petomimetics can target cancercells, in particular cells that exhibit an I_(v)l₃ integrin. Thus, onecould use RGD peptides, cyclic peptides containing RGD, RGD peptidesthat include D-amino acids, as well as synthetic RGD mimics. In additionto RGD, one can use other moieties that target the I_(v)-9³ integrinligand. Generally, such ligands can be used to control proliferatingcells and angiogeneis. Preferred conjugates of this type include an iRNAagent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancergene described herein.

The iRNA agents of the invention are particularly useful when targetedto the liver. An iRNA agent can be targeted to the liver byincorporation of a monomore derivitzed with a ligand which targets tothe liver. For example, a liver-targeting agent can be a lipophilicmoiety. Preferred lipophilic moieties include lipid, cholesterols,oleyl, retinyl, or cholesteryl residues. Other lipophilic moieties thatcan function as liver-targeting agents include cholic acid, adamantaneacetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine.

An iRNA agent can also be targeted to the liver by association with alow-density lipoprotein (LDL), such as lactosylated LDL. Polymericcarriers complexed with sugar residues can also function to target iRNAagents to the liver.

A targeting agent that incorporates a sugar, e.g., galactose and/oranalogues thereof, is particularly useful. These agents target, inparticular, the parenchymal cells of the liver. For example, a targetingmoiety can include more than one or preferably two or three galactosemoieties, spaced about 15 angstroms from each other. The targetingmoiety can alternatively be lactose (e.g., three lactose moieties),which is glucose coupled to a galactose. The targeting moiety can alsobe N-Acetyl-Galactosamine, N—Ac-Glucosamine A mannose ormannose-6-phosphate targeting moiety can be used for macrophagetargeting.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long (see Table 1, forexample).

TABLE 1  Exemplary Cell Permeation Peptides Cell Permeation PeptideAmino acid Sequence Reference Penetratin RQIKIWFQNRRMKWKK (SEQ ID NO: 1)Derossi et al., J. Biol. Chem. 269:10444, 1994 Tat fragmentGRKKRRQRRRPPQC (SEQ ID NO: 2) Vives et al., J. (48-60)Biol. Chem., 272:16010, 1997 Signal Sequence-GALFLGWLGAAGSTMGAWSQPKKKRKV Chaloin et al., based peptide (SEQ ID NO: 3)Biochem. Biophys. Res. Commun., 243:601, 1998 PVECLLIILRRRIRKQAHAHSK (SEQ ID NO: 4) Elmquist et al.,Exp. Cell Res., 269:237, 2001 Transportan GWTLNSAGYLLKINLKALAALAKKILPooga et al., (SEQ ID NO: 5) FASEB J., 12:67, 1998 AmphiphilicKLALKLALKALKAALKLA (SEQ ID NO: 6) Oehlke et al., model peptideMol. Ther., 2:339, 2000 Arg₉ RRRRRRRRR (SEQ ID NO: 7)Mitchell et al., J. Pept. Res., 56:318, 2000 Bacterial cellKFFKFFKFFK (SEQ ID NO: 8) wall permeating LL-37LLGDFFRKSKEKIGKEFKRIVQRIKDFLRN LVPRTES (SEQ ID NO: 9) Cecropin P1SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (SEQ ID NO: 10) α-defensinACYCRIPACIAGERRYGTCIYQGRLWAFCC (SEQ ID NO: 11) b-defensinDHYNCVSSGGQCLYSACPIFTKIQGTCYR GKAKCCK (SEQ ID NO: 12) BactenecinRKCRIVVIRVCR (SEQ ID NO: 13) PR-39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (SEQ ID NO: 14) IndolicidinILPWKWPWWPWRR-NH2 (SEQ ID NO: 15)

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO:16). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO:17)) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:18)) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:19))have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Preferably the peptide or peptidomimetic tethered toan iRNA agent via an incorporated monomer unit is a cell targetingpeptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGDmimic. A peptide moiety can range in length from about 5 amino acids toabout 40 amino acids. The peptide moieties can have a structuralmodification, such as to increase stability or direct conformationalproperties. Any of the structural modifications described below can beutilized.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingof an iRNA agent to tumors of a variety of other tissues, including thelung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy8:783-787, 2001). Preferably, the RGD peptide will facilitate targetingof an iRNA agent to the kidney. The RGD peptide can be linear or cyclic,and can be modified, e.g., glycosylated or methylated to facilitatetargeting to specific tissues. For example, a glycosylated RGD peptidecan deliver an iRNA agent to a tumor cell expressing a_(v)β₃ (Haubner etal., Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an I_(v)l₃ integrin.Thus, one could use RGD peptides, cyclic peptides containing RGD, RGDpeptides that include D-amino acids, as well as synthetic RGD mimics. Inaddition to RGD, one can use other moieties that target the I_(v)-l₃integrin ligand. Generally, such ligands can be used to controlproliferating cells and angiogeneis. Preferred conjugates of this typeinclude an iRNA agent that targets PECAM-1, VEGF, or other cancer gene,e.g., a cancer gene described herein.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide tethered to an ligand-conjugatedmonomer can be an amphipathic α-helical peptide. Exemplary amphipathicα-helical peptides include, but are not limited to, cecropins,lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP),cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinalantimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins,melittins, pleurocidin, H₂A peptides, Xenopus peptides, esculentinis-1,and caerins. A number of factors will preferably be considered tomaintain the integrity of helix stability. For example, a maximum numberof helix stabilization residues will be utilized (e.g., leu, ala, orlys), and a minimum number helix destabilization residues will beutilized (e.g., proline, or cyclic monomeric units. The capping residuewill be considered (for example Gly is an exemplary N-capping residueand/or C-terminal amidation can be used to provide an extra H-bond tostabilize the helix. Formation of salt bridges between residues withopposite charges, separated by i±3, or i±4 positions can providestability. For example, cationic residues such as lysine, arginine,homo-arginine, ornithine or histidine can form salt bridges with theanionic residues glutamate or aspartate.

Peptide and petidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; a, 13, or ypeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

In some embodiments, the ligand can be any of the nucleobases describedherein.

In some embodiments, the ligand can be a substituted amine, e.g.dimethylamino. In certain embodiments the substituted amine can berendered cationic, e.g., by quaternization, e.g., protonation oralkylation. In certain embodiments, the substituted amine can be at theterminal position of a relatively hydrophobic chain, e.g., an alkylenechain.

In some embodiments, the ligand can be one of the following triterpenes:

In some embodiments, the ligand can be substituted or unsubstitutedcholesterol, or a stereoisomer thereof or one of the following steroids:

Methods for Making IRNA Agents

A listing of ribonucleosides containing the unusual bases describedherein are described in “The RNA Modification Database” maintained byPamela F. Crain, Jet Rozenski and James A. McCloskey; Departments ofMedicinal Chemistry and Biochemistry, University of Utah, Salt LakeCity, Utah 84112, USA (RNAmods@lib.med.utah.edu)

The 5′ silyl protecting group can be used in conjunction with acidlabile orthoesters at the 2′ position of ribonucleosides to synthesizeoligonucleotides via phosphoramidite chemistry. Final deprotectionconditions are known not to significantly degrade RNA products.Functional groups on the unusual and universal bases are blocked duringoligonucleotide synthesis with protecting groups that are compatiblewith the operations being performed that are described herein. Allsyntheses can be can be conducted in any automated or manual synthesizeron large, medium, or small scale. The syntheses may also be carried outin multiple well plates or glass slides.

The 5′-O-silyl group can be removed via exposure to fluoride ions, whichcan include any source of fluoride ion, e.g., those salts containingfluoride ion paired with inorganic counterions e.g., cesium fluoride andpotassium fluoride or those salts containing fluoride ion paired with anorganic counterion, e.g., a tetraalkylammonium fluoride. A crown ethercatalyst can be utilized in combination with the inorganic fluoride inthe deprotection reaction. Preferred fluoride ion source aretetrabutylammonium fluoride or aminehydrofluorides (e.g., combiningaqueous HF with triethylamine in a dipolar aprotic solvent, e.g.,dimethylformamide).

The choice of protecting groups for use on the phosphite triesters andphosphotriesters can alter the stability of the triesters towardsfluoride. Methyl protection of the phosphotriester or phosphitetriestercan stabilize the linkage against fluoride ions and improve processyields.

Since ribonucleosides have a reactive 2′ hydroxyl substituent, it can bedesirable to protect the reactive 2′ position in RNA with a protectinggroup that is compatible with a 5′-O-silyl protecting group, e.g. onestable to fluoride. Orthoesters meet this criterion and can be readilyremoved in a final acid deprotection step that can result in minimal RNAdegradation.

Tetrazole catalysts can be used in the standard phosphoramidite couplingreaction. Preferred catalysts include e.g. tetrazole, S-ethyl-tetrazole,p-nitrophenyltetrazole.

The general process is as follows. Nucleosides are suitably protectedand functionalized for use in solid-phase or solution-phase synthesis ofRNA oligonucleotides. The 2′-hydroxyl group in a ribonucleotide can bemodified using a tris orthoester reagent. The 2′-hydroxyl can bemodified to yield a 2′-O-orthoester nucleoside by reacting theribonucleoside with the tris orthoester reagent in the presence of anacidic catalyst, e.g., pyridinium p-toluene sulfonate.

This reaction is known to those skilled in the art. The product can thenbe subjected to further protecting group reactions (e.g.,5′-O-silylation) and functionalizations (e.g., 3′-β-phosphitylation) toproduce a desired reagent (e.g., nucleoside phosphoramidite) forincorporation within an oligonucleotide or polymer by reactions known tothose skilled in the art.

Preferred orthoesters include those comprising ethylene glycol ligandswhich are protected with acyl or ester protecting groups. Specifically,the preferred acyl group is acetyl. The nucleoside reagents may then beused by those skilled in the art to synthesize RNA oligonucleotides oncommercially available synthesizer instruments, e.g. Gene Assembler Plus(Pharmacia), 380B (Applied Biosystems). Following synthesis (eithersolution-phase or solid-phase) of an oligonucleotide or polymer, theproduct can be subjected to one or more reactions using non-acidicreagents. One of these reactions may be strong basic conditions, forexample, 40% methylamine in water for 10 minutes at 55.degree. C., whichwill remove the acyl protecting groups from the ethylene glycol ligandsbut leave the orthoester moiety attached. The resultant orthoester maybe left attached when the polymer or oligonucleotide is used insubsequent applications, or it may be removed in a final mildly-acidicreaction, for example, 10 minutes at 55.degree. C. in 50 mM acetic acid,pH 3.0, followed by addition of equal volume of 150 mM TRIS buffer for10 minutes at 55.degree. C.

Universal bases are described in “Survey and Summary: The Applicationsof Universal DNA base analogues” Loakes, D., Nucleic Acid Research 2001,29, 2437, which is incorporated by reference in its entirety. Specificexamples are described in the following: Liu, D.; Moran, S.; Kool, E. T.Chem. Biol., 1997, 4, 919-926; Morales, J. C.; Kool, E. T. Biochemistry,2000, 39, 2626-2632; Matray, T, J.; Kool, E. T. J. Am. Chem. Soc., 1998,120, 6191-6192; Moran, S. Ren, R. X.-F.; Rumney I V, S.; Kool, E. T. J.Am. Chem. Soc., 1997, 119, 2056-2057; Guckian, K. M.; Morales, J. C.;Kool, E. T. J. Org. Chem., 1998, 63, 9652-9656; Berger, M.; Wu. Y.;Ogawa, A. K.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. NucleicAcids Res., 2000, 28, 2911-2914; Ogawa, A. K.; Wu, Y.; McMinn, D. L.;Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122,3274-3287; Ogawa, A. K.; Wu. Y.; Berger, M.; Schultz, P. G.; Romesberg,F. E. J. Am. Chem. Soc., 2000, 122, 8803-8804; Tae, E. L.; Wu, Y.; Xia,G.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2001, 123,7439-7440; Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz, P.G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 7621-7632;. McMinn,D. L.; Ogawa. A. K.; Wu, Y.; Liu, J.; Schultz, P. G.; Romesberg, F. E.J. Am. Chem. Soc., 1999, 121, 11585-11586; Brotschi, C.; Haberli, A.;Leumann, C, J. Angew. Chem. Int. Ed., 2001, 40, 3012-3014; Weizman, H.;Tor, Y. J. Am. Chem. Soc., 2001, 123, 3375-3376; Lan, T.; McLaughlin, L.W. J. Am. Chem. Soc., 2000, 122, 6512-13.

As discussed above, the monomers and methods described herein can beused in the preparation of modified RNA molecules, or polymericmolecules comprising any combination of monomer compounds describedherein and/or natural or modified ribonucleotides in which one or moresubunits contain an unusual or universal base. Modified RNA moleculesinclude e.g. those molecules containing a chemically or stereochemicallymodified nucleoside (e.g., having one or more backbone modifications,e.g., phosphorothioate or P-alkyl; having one or more sugarmodifications, e.g., 2′-OCH₃ or 2′-F; and/or having one or more basemodifications, e.g., 5-alkylamino or 5-allylamino) or a nucleosidesurrogate.

Coupling of 5′-hydroxyl groups with phosphoramidites forms phosphiteester intermediates, which in turn are oxidized e.g., with iodine, tothe phosphate diester. Alternatively, the phosphites may be treated withe.g., sulfur, selenium, amino, and boron reagents to form modifiedphosphate backbones. Linkages between the monomers described herein anda nucleoside or oligonucleotide chain can also be treated with iodine,sulfur, selenium, amino, and boron reagents to form unmodified andmodified phosphate backbones respectively. Similarly, the monomersdescribed herein may be coupled with nucleosides or oligonucleotidescontaining any of the modifications or nucleoside surrogates describedherein.

The synthesis and purification of oligonucleotide peptide conjugates canbe performed by established methods. See, for example, Trufert et al.,Tetrahedron, 52:3005, 1996; and Manoharan, “Oligonucleotide Conjugatesin Antisense Technology,” in Antisense Drug Technology, ed. S. T.Crooke, Marcel Dekker, Inc., 2001. Exemplary methods are shown in FIGS.4 and 5.

In one embodiment of the invention, a peptidomimetic can be modified tocreate a constrained peptide that adopts a distinct and specificpreferred conformation, which can increase the potency and selectivityof the peptide. For example, the constrained peptide can be anazapeptide (Gante, Synthesis, 405-413, 1989). An azapeptide issynthesized by replacing the α-carbon of an amino acid with a nitrogenatom without changing the structure of the amino acid side chain. Forexample, the azapeptide can be synthesized by using hydrazine intraditional peptide synthesis coupling methods, such as by reactinghydrazine with a “carbonyl donor,” e.g., phenylchloroformate. A generalazapeptide synthesis is shown in FIG. 6.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to an ligand-conjugated monomer) canbe an N-methyl peptide. N-methyl peptides are composed of N-methyl aminoacids, which provide an additional methyl group in the peptide backbone,thereby potentially providing additional means of resistance toproteolytic cleavage. N-methyl peptides can by synthesized by methodsknown in the art (see, for example, Lindgren et al., Trends Pharmacol.Sci. 21:99, 2000; Cell Penetrating Peptides: Processes and Applications,Langel, ed., CRC Press, Boca Raton, Fla., 2002; Fische et al.,Bioconjugate. Chem. 12: 825, 2001; Wander et al., J. Am. Chem. Soc.,124:13382, 2002). For example, an Ant or Tat peptide can be an N-methylpeptide. An exemplary synthesis is shown in FIG. 7.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to a ligand-conjugated monomer) canbe a β-peptide. β-peptides form stable secondary structures such ashelices, pleated sheets, turns and hairpins in solutions. Their cyclicderivatives can fold into nanotubes in the solid state. β-peptides areresistant to degradation by proteolytic enzymes. β-peptides can besynthesized by methods known in the art. For example, an Ant or Tatpeptide can be a β-peptide. An exemplary synthesis is shown in FIG. 8.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to a ligand-conjugated monomer) canbe a oligocarbamate. Oligocarbamate peptides are internalized into acell by a transport pathway facilitated by carbamate transporters. Forexample, an Ant or Tat peptide can be an oligocarbamate. An exemplarysynthesis is shown in FIG. 9.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to a ligand-conjugated monomer) canbe an oligourea conjugate (or an oligothiourea conjugate), in which theamide bond of a peptidomimetic is replaced with a urea moiety.Replacement of the amide bond provides increased resistance todegradation by proteolytic enzymes, e.g., proteolytic enzymes in thegastrointestinal tract. In one embodiment, an oligourea conjugate istethered to an iRNA agent for use in oral delivery. The backbone in eachrepeating unit of an oligourea peptidomimetic can be extended by onecarbon atom in comparison with the natural amino acid. The single carbonatom extension can increase peptide stability and lipophilicity, forexample. An oligourea peptide can therefore be advantageous when an iRNAagent is directed for passage through a bacterial cell wall, or when aniRNA agent must traverse the blood-brain barrier, such as for thetreatment of a neurological disorder. In one embodiment, a hydrogenbonding unit is conjugated to the oligourea peptide, such as to createan increased affinity with a receptor. For example, an Ant or Tatpeptide can be an oligourea conjugate (or an oligothiourea conjugate).An exemplary synthesis is shown in FIG. 10.

The siRNA peptide conjugates of the invention can be affiliated with,e.g., tethered to, ligand-conjugated monomers occurring at variouspositions on an iRNA agent. For example, a peptide can be terminallyconjugated, on either the sense or the antisense strand, or a peptidecan be bisconjugated (one peptide tethered to each end, one conjugatedto the sense strand, and one conjugated to the antisense strand). Inanother option, the peptide can be internally conjugated, such as in theloop of a short hairpin iRNA agent. In yet another option, the peptidecan be affiliated with a complex, such as a peptide-carrier complex.

A peptide-carrier complex consists of at least a carrier molecule, whichcan encapsulate one or more iRNA agents (such as for delivery to abiological system and/or a cell), and a peptide moiety tethered to theoutside of the carrier molecule, such as for targeting the carriercomplex to a particular tissue or cell type. A carrier complex can carryadditional targeting molecules on the exterior of the complex, orfusogenic agents to aid in cell delivery. The one or more iRNA agentsencapsulated within the carrier can be conjugated to lipophilicmolecules, which can aid in the delivery of the agents to the interiorof the carrier.

A carrier molecule or structure can be, for example, a micelle, aliposome (e.g., a cationic liposome), a nanoparticle, a microsphere, ora biodegradable polymer. A peptide moiety can be tethered to the carriermolecule by a variety of linkages, such as a disulfide linkage, an acidlabile linkage, a peptide-based linkage, an oxyamino linkage or ahydrazine linkage. For example, a peptide-based linkage can be a GFLGpeptide. Certain linkages will have particular advantages, and theadvantages (or disadvantages) can be considered depending on the tissuetarget or intended use. For example, peptide based linkages are stablein the blood stream but are susceptible to enzymatic cleavage in thelysosomes. A schematic of preferred carriers is shown in FIG. 11.

The protected monomer compounds can be separated from a reaction mixtureand further purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. Other synthetic chemistry transformations, protecting groups(e.g., for hydroxyl, amino, etc. present on the bases) and protectinggroup methodologies (protection and deprotection) useful in synthesizingthe compounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The protected monomer compounds of this invention may contain one ormore asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of these compounds areexpressly included in the present invention. The compounds describedherein can also contain linkages (e.g., carbon-carbon bonds,carbon-nitrogen bonds, e.g., amides) or substituents that can restrictbond rotation, e.g. restriction resulting from the presence of a ring ordouble bond. Accordingly, all cis/trans, E/Z isomers, and rotationalisomers (rotamers) are expressly included herein. The compounds of thisinvention may also be represented in multiple tautomeric forms, in suchinstances, the invention expressly includes all tautomeric forms of thecompounds described herein (e.g., alkylation of a ring system may resultin alkylation at multiple sites, the invention expressly includes allsuch reaction products). All such isomeric forms of such compounds areexpressly included in the present invention. All crystal forms of thecompounds described herein are expressly included in the presentinvention. Representative ligand-conjugated monomers and typicalsyntheses for preparing ligand-conjugated monomers and related compoundsdescribed herein are provided below. As discussed elsewhere, protectinggroups for ligand-conjugated monomer hydroxyl groups, e.g., OFG¹,include but are not limited to the dimethoxytrityl group (DMT). Forexample, it can be desirable in some embodiments to use silicon-basedprotecting groups as a protecting group for OFG¹. Silicon-basedprotecting groups can therefore be used in conjunction with or in placeof the DMT group as necessary or desired. Thus, the ligand-conjugatedmonomers and syntheses delineated below, which feature the DMTprotecting group as a protecting group for OFG¹, is not to be construedas limiting in any way to the invention.

Synthesis of Pyrroline Earrier

Synthesis of 5′-Labelled siRNA

25 & 26 can be used for 3′,5′-conjugation respectively.

Synthesis of Pthalimido Derivative

30 and 31 can be converted to similar derivatives as shown in schemes2-4 for 3′ and 5′ cpnjugation of si RNA

Synthesis of Thalimido Derivative

40 and 41 can be converted to similar derivatives as shown in schemes2-4 for 3′ and 5′ cpnjugation of siRNA

Synthesis of N-Alkyl Pyrroline Derivatives

Intermediates 50 and 51 can be converted to analogs which could beconjugated with siRNA using similar reactions

Piperidine Series Ligands:

Similar to pyrroline series piperidine series can be synthesised

Piperidine Series Ligands:

Similar to pyrroline series piperidine series can be synthesised

Hydroxy Proline Series Linkers:

From commercially available cis-3-hydroxy proline and (s)-pyrrolidonecarboxylate

Phthalimide Derivative to Stabilise siRNA

4-Hydroxy Proline Derivatives

Phthalimido Derivatives

Synthesis of 6-Membered Linker

Similar reaction can be carried out with 2-piperidone and 3-piperidone

Linkers from 4-Piperidone

Linkers from 3-Piperidone

Linkers from 2-Piperidone

Conjugation Through Decalin System

Conjugates from Decalin System:

Decalin Linker from Wieland-Miescher Ketone

Conjugates from Wieland-Miescher Ketone

Synthesis of Pyrroline Linker

Solid Phase Synthesis and Post-Synthesis Conjugation:

Exemplary Ligand Conjugated Monomers

LCM-E.g.-

Targeting

The iRNA agents of the invention are particularly useful when targetedto the liver. The chemical modifications described herein can becombined with the compounds and methods described in U.S. ProvisionalApplication 60/462,097, filed on Apr. 9, 2003, which is herebyincorporated by reference; and U.S. Provisional Application 60/461,915,filed on Apr. 10, 2003, which is hereby incorporated by reference. Forexample, an iRNA agent can be targeted to the liver by incorporation ofan RRMS containing a ligand that targets the liver, e.g., a lipophilicmoiety. Preferred lipophilic moieties include lipid, cholesterols,oleyl, retinyl, or cholesteryl residues. Other lipophilic moieties thatcan function as liver-targeting agents include cholic acid, adamantaneacetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine.

An iRNA agent can also be targeted to the liver by association with alow-density lipoprotein (LDL), such as lactosylated LDL. Polymericcarriers complexed with sugar residues can also function to target iRNAagents to the liver.

Conjugation of an iRNA agent with a serum albumin (SA), such as humanserum albumin, can also be used to target the iRNA agent to a non-kidneytissue, such as the liver.

An iRNA agent targeted to the liver by an RRMS targeting moietydescribed herein can target a gene expressed in the liver. For example,the iRNA agent can target p21(WAF1/DIP1), P27(KIP1), the α-fetoproteingene, beta-catenin, or c-MET, such as for treating a cancer of theliver. In another embodiment, the iRNA agent can target apoB-100, suchas for the treatment of an HDL/LDL cholesterol imbalance; dyslipidemias,e.g., familial combined hyperlipidemia (FCHL), or acquiredhyperlipidemia; hypercholesterolemia; statin-resistanthypercholesterolemia; coronary artery disease (CAD); coronary heartdisease (CHD); or atherosclerosis. In another embodiment, the iRNA agentcan target forkhead homologue in rhabdomyosarcoma (FKHR); glucagon;glucagon receptor; glycogen phosphorylase; PPAR-Gamma Coactivator(PGC-1); Fructose-1,6-bisphosphatase; glucose-6-phosphatase;glucose-6-phosphate translocator; glucokinase inhibitory regulatoryprotein; or phosphoenolpyruvate carboxykinase (PEPCK), such as toinhibit hepatic glucose production in a mammal, such as a human, such asfor the treatment of diabetes. In another embodiment, an iRNA agenttargeted to the liver can target Factor V, e.g., the Leiden Factor Vallele, such as to reduce the tendency to form a blood clot. An iRNAagent targeted to the liver can include a sequence which targetshepatitis virus (e.g., Hepatitis A, B, C, D, E, F, G, or H). Forexample, an iRNA agent of the invention can target any one of thenonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatmentof hepatitis B, an iRNA agent can target the protein X (HBx) gene, forexample.

A targeting agent that incorporates a sugar, e.g., galactose and/oranalogues thereof, can be useful. These agents target, for example, theparenchymal cells of the liver. For example, a targeting moiety caninclude more than one or preferably two or three galactose moieties,spaced about 15 angstroms from each other. The targeting moiety canalternatively be lactose (e.g., three lactose moieties), which isglucose coupled to a galactose. The targeting moiety can also beN-Acetyl-Galactosamine, N—Ac-Glucosamine A mannose ormannose-6-phosphate targeting moiety can be used for macrophagetargeting.

The iRNA agents of the invention are particularly useful when targetedto the kidney. The chemical modifications described herein can becombined with the compounds and methods described in U.S. ProvisionalApplication 60/460,783, filed on Apr. 3, 2003, which is herebyincorporated by reference; and 60/503,414, filed on Sep. 15, 2003, whichis hereby incorporated by reference. An iRNA agent can be targeted tothe kidney by incorporation of an RRMS containing a ligand that targetsthe kidney.

An iRNA agent targeted to the kidney by an RRMS targeting moietydescribed herein can target a gene expressed in the kidney.

Ligands on RRMSs can include folic acid, glucose, cholesterol, cholicacid, Vitamin E, Vitamin K, or Vitamin A.

Conjugation with a Lipophilic Moiety which Promotes Entry into Cells

RNAi agents can be modified so as to enhance entry into cells, e.g., byconjugation with a lipophilic moiety. A lipophilic moiety can beattached to an RNAi agent in a number of ways but a preferred mode ofattachment is by attachment to an RRMS, e.g., pyrroline-based RRMS. Thelipohilic moiety can be attached at the N atom of a pyrroline-basedRRMS. Examples of lipophilic moieties include cholesterols, lipid,oleyl, retinyl, or cholesteryl residues. Other lipophilic moietiesinclude cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. Cholesterolis a particularly preferred example.

The lipohilic moiety can be attached at the 3′ terminus, the 5′terminus, or internally, preferably on the sense strand. The lipohilicmoiety can be attached to an RRMS, e.g., a pyrroline-based RRMS which isat the 3′ terminus, the 5′ terminus, or internal, in the sense strand.The attachment can be direct or through a tethering molecule. Tethers,spacers or linkers discussed herein can be used to attach the moiety tothe RRMS.

An iRNA agent to which one or more lipophilic (e.g., cholesterol)molecules is conjugated (referred to herein as an “iRNA-lipophilicconjugate”) can be delivered in vivo, e.g., to a cell, such as a cell ofa tissue in a subject, such as a mammalian subject (e.g., a human ormouse). Alternatively, or in addition, the iRNA agent can be deliveredin vitro, e.g., to a cell in a cell line. Cell lines can be, forexample, from a vertebrate organism, such as a mammal (e.g., a human ora mouse). Delivery of an iRNA-cholesterol conjugate to a cell line canbe in the absence of other transfection reagents. For example, deliveryof an iRNA-lipophilic conjugate to a cell can be in the absence of, oroptionally, in the presence of, Lipofectamine™ (Invitrogen, Carlsbad,Calif.), Lipofectamine 2000™, TranslT-TKO™ (Mirus, Madison, Wis.),FuGENE 6 (Roche, Indianapolis, Ind.), polyethylenimine, X-tremeGENE Q2(Roche, Indianapolis, Ind.), DOTAP, DOSPER, or Metafectene™ (Biontex,Munich, Germany), or another transfection reagent. Exemplary cell linescan be provided by the American Type Culture Collection (ATCC)(Manassus, Va.). An iRNA-lipophilic conjugate can be delivered to a cellline, such as any cell line described herein, to target a specific genefor downregulation.

In one example, an iRNA-lipophilic conjugate can be delivered to aprimary cell line, e.g., a synoviocyte (such as type B), cardiacmyocyte, keratinocyte, hepatocyte, smooth muscle cell, endothelial cell,or dermal fibroblast cell line.

In another example, an iRNA-lipophilic conjugate can be delivered tomonocyte, or myeloid cell line, e.g., a THP1, Raw264.7, IC₂₁, P388D1,U937, or HL60 cell line.

In another example, an iRNA-lipophilic conjugate can be delivered tolymphoma, or leukemia cell line, e.g., an SEM-K2, WEHI-231, HB56, TIB55,Jurkat, K562, EL4, LRMB, Bel-1, or TF1 cell line. For example, aniRNA-lipophilic conjugate can be delivered to a lymphoma cell line totarget a specific gene for down regulation. An iRNA-lipophilic agent cantarget (down-regulate) a gene in a Jurkat cell line, for example, thatencodes an immune factor, such as an interleukin gene, e.g., IL-1, IL-2,IL-5, IL-6, IL-8, IL-10, IL-15, IL-16, IL-17, or IL-18. In anotheraspect, an iRNA-lipophilic conjugate can target a gene that encodes areceptor of an interleukin.

An iRNA-lipophilic conjugate can target a gene resulting from achromosomal translocation, such as BCR-ABL, TEL-AML-1, EWS-FLI1,EWS-ERG, TLS-FUS, PAX3-FKHR, or AML1-ETO. For example, aniRNA-lipophilic conjugate that targets a gene resulting from achromosomal translocation can be delivered to a leukemia cell line,e.g., any of the leukemia cell lines discussed above.

An iRNA-lipophilic conjugate can be delivered to an immortalized cellline, including immortalized cell lines from a variety of differenttissue types, including but not limited to T-cells, fibroblast cells,epithelial cells (e.g., kidney epithelial cells) and muscle cells (e.g.,smooth muscle cells). Exemplary immortalized cell lines are CTLL-2(T-cell), Rat 6 (fibroblast), VERO (fibroblast), MRCS (fibroblast), CV1(fibroblast), Cos₇ (fibroblast), RPTE (kidney epithelial), and A10(smooth muscle) cell lines.

An iRNA-lipophilic conjugate can be delivered to a mast cell line, forexample. An iRNA-lipophilic conjugate delivered to a mast cell line cantarget, for example, a gene encoding a GRB2 associated binding protein(e.g., GAB2).

An iRNA-lipophilic conjugate can be delivered to an adherent tumor cellline, including tumor cell lines from a variety of different tissuetypes including but not limited to cancers of the bladder, lung, breast,cervix, colon, pancreas, prostate, and liver, melanomas, andglioblastomas. Exemplary tumor cell lines include the T24 (bladder), J82(bladder), A549 (lung), Calul (lung), SW480 (colon), SW620 (colon),CaCo2 (colon), A375 (melanoma), C8161 (melanoma), MCF-7 (breast),MDA-MB-231 (breast), HeLa (cervical), HeLa S3 (cervical), MiaPaCall(pancreas), Panel (pancreas), PC-3 (prostate), LNCaP (prostate), HepG2(hepatocellular), and U87 (glioblastoma) cell lines. An iRNA-lipophilicconjugate that targets a specific gene can be delivered to an adherenttumor cell line. For example, an iRNA-lipophilic conjugate that targetsa growth factor or growth factor receptor, such as a TGF-beta (e.g.,TGF-beta 1) or TGF-beta receptor gene, can be delivered to an A549 orHepG2 cell line, a DLD2 colon carcinoma line, or a SKOV3 adenocarcinomacell line. Other exemplary target growth factor genes include plateletderived growth factor (PDGF) and PDGF-Receptor (PDGFR), vascularendothelial growth factor (VEGF) and VEGF receptor genes (e.g., VEGFr1,VEGFr2, or VEGFr3), and insulin-growth factor receptors, such as type Iinsulin-growth factor (IGF) receptors, including IGF-1R, DAF-2 and 1nR.

In another example, an iRNA-lipophilic conjugate that targets one ormore genes in a protein tyrosine phosphatase type IVA (PRL3, also calledPTP4A3) gene family (e.g., PRL1, PRL2, or PRL3), or a gene in a PRL3pathway, can be delivered to an A549 cell line, or to a culturedcolorectal epithelial cell line.

In another example, an iRNA-lipophilic conjugate can target one or moreprotein kinase C genes in an adherent tumor cell line, such as in amouse Lewis lung carcinoma, B16 melanoma, mouse mammary adenocarcinomaor fibrosarcoma; or a human lung carcinoma, bladder carcinoma,pancreatic cancer, gastric cancer, breast cancer, thyroid carcinoma, ormelanoma. An iRNA-lipophilic conjugate can target a gene encoding a PKCisoforms, such as PKC-alpha, PKC beta I, PKC beta II, PKC gamma, PKCdelta, PKC epsilon, and/or PKC zeta, or a gene encoding one or morereceptors of a protein kinase C polypeptide.

In another example, an iRNA-lipophilic conjugate can target a geneencoding a P-glycoprotein, such as a gene in the multidrug resistance(MDR) gene family, e.g., MDR1. An iRNA-lipophilic conjugate that targetsan MDR gene can be delivered, for example, to a human KB carcinoma cellline, a human leukemia or ovarian carcinoma cell line, or a lungcarcinoma cell line such as A549.

In another example, an iRNA-lipophilic conjugate can target a geneencoding a gene in the telomerase pathway, such as TERT or thetelomerase template RNA (TR/TERC). An iRNA-lipophilic conjugate thattargets a gene in the telomerase pathway can be delivered, for example,to a human cancer cell line, e.g., a breast, cervical, endometrial,meningeal, lung, testicular, or ovarian cancer cell line.

In another example, an iRNA-lipophilic conjugate delivered to anadherent cell line (e.g., a HeLa, parathyroid adenoma, or A549 cellline) can target a cyclin gene, such as cyclin Dl.

In another example, an iRNA-lipophilic conjugate delivered to anadherent cell line (e.g., a HeLa cell line) can target an NF-kappaB orREL-A gene, or a gene encoding a ligand or receptor of an NF-kappaB orREL-A polypeptide, or a gene encoding a subunit of NF-kappaB, such asREL-B, REL, NF-kappaB1 or NF-kappaB2.

In another example, an iRNA-lipophilic conjugate delivered to anadherent cell line (e.g., a HeLa or A549 cell line) can target a geneencoding proliferating cell nuclear antigen (PCNA), a checkpoint kinasegene (CHK-1), or a c-fos gene. Further, an iRNA-lipophilic conjugate cantarget any gene in a PCNA, CHK-1, or c-fos pathway. For example aniRNA-lipophilic conjugate can down-regulate a gene encoding jun, whichis in the c-fos pathway.

In another example, an iRNA-lipophilic conjugate delivered to anadherent cell line (e.g., an A549, T24, or A375 cell line) can target agene encoding BCL2.

The cell lines described herein can be used to test iRNA-lipophilicconjugates that target exogenous, such as pathogenic or viral, nucleicacids. For example, an iRNA-lipophilic conjugate that targets ahepatitis viral gene can be delivered to a human hepatoma cell line,such as a HepG2 or Huh cell line, e.g., Huh1, Huh4, Huh7, and the like,that has been infected with the virus (e.g., an HAV, HBV, or HCV). Forexample, an iRNA-lipophilic conjugate that targets an HCV gene, such asin an infected Huh cell line, can target a conserved region of the HCVgenome, such as the 5′-non-coding region (NCR), the 5′ end of the coreprotein coding region, or the 3′-NCR.

The cell lines described herein can be also be used to testiRNA-lipophilic conjugates that target exogenous recombinant nucleicacids, such as reporter genes (e.g., GFP, lacZ, beta-galactosidase, andthe like), that are transfected (transiently or stably) into the celllines.

In one aspect, an iRNA-lipophilic conjugate can be delivered to a B-cellline, e.g., BC-3, C1R, or ARH-77 cells. In another aspect, aniRNA-lipophilic conjugate can be delivered to T-cells, e.g., J45.01,MOLT, and CCRF-CEM cells. An iRNA-lipophilic conjugate can target anendogenous or exogenous nucleic acid. For example, development of aniRNA-lipophilic conjugate that targets an HIV gene can be tested againstan exogenous HIV nucleic acid in a B cell or T cell line, or in amacrophage or endothelial cell culture system.

An iRNA-lipophilic conjugate can be delivered to cells derived fromendoderm, epithelium, or mesoderm. For example, an iRNA-lipophilicconjugate can be delivered to cells of the HeLa or MCF7 epithelial celllines, to cells of the HUVEC endothelial cell line, or to cells of anSK-UT or HASMC mesodermal cell line. In one example, an iRNA-lipophilicagent that targets a TGF-beta nucleic acid or TGF-beta receptor nucleicacid can be delivered to a vascular smooth muscle cell line, e.g., thekidney fibroblast 293 cell line. Other exemplary targets ofiRNA-lipophilic conjugates delivered to fibroblast cells, such as 293cells, included a protein tyrosine phosphatase-1B (PTP-1B) gene or MAPkinase gene (e.g., ERK1, ERK2, JNK1, JNK2, and p38). In another example,an iRNA-lipophilic conjugate that targets an MDR gene fordown-regulation can be delivered to the human intestinal epithelial cellline, Caco-2.

In one example, an iRNA-lipophilic conjugate delivered to a cell line,such as an epithelial or mesodermal cell line (e.g., a HeLa or HASMCcell line, respectively), can target a gene encoding a Myc or Mybpolypeptide, e.g., c-Myc, N-Myc, L-Myc, c-Myb, a-Myb, b-Myb, and v-Myb,or a gene in the Myc or Myb gene pathway, such as cyclin D1, cyclin D2,cyclin E, CDK4, cdc25A, CDK2, or CDK4.

In one example, an iRNA-lipophilic conjugate that targets a geneexpressed in the nervous system, such as in the brain, e.g, a G72 orD-amino acid oxidase (DAAO) gene, can be delivered to a culturedneuronal cell line, such as an hNT cell line.

In another example, an iRNA-lipophilic conjugate can target a geneencoding a gene in the telomerase pathway, such as TERT or TR/TERC. AniRNA-lipophilic conjugate that targets a gene in the telomerase pathwaycan be delivered, for example, to a human keratinocyte cell line, suchas a HEK cell line, e.g., HEKn or HEKa.

In another example, an iRNA-lipophilic conjugate delivered to atissue-specific cell-line, such as a HEK (keratinocyte), HuVEC(endothelial), 3T3 (fibroblast), or NHDF (fibroblast) cell line, cantarget a gene encoding BCL-2, or VEGF or a VEGF receptor (e.g., VEGFr1,VEGFr2, or VEGFr3).

An iRNA-lipophilic conjugate can be delivered to a subgroup of cellsderived from a particular tissue. For example, an iRNA-lipophilicconjugate can be delivered to a proximal tubular kidney cell line, suchas the mouse cell line mIMCD-3. An iRNA-lipophilic conjugate thattargets a TGF-beta nucleic acid or TGF-beta receptor nucleic acid, forexample, can be delivered to a cell line derived from prostate tissue,e.g., a PC3 or RWPE prostate cell line. An iRNA-lipophilic conjugatedelivered to a prostate tissue cell line can alternatively target apolycomb group gene, such as EZH2.

In another example, an iRNA-lipophilic conjugate can be delivered topancreatic islet b-cells, where for example, it targets a gastricinhibitory polypeptide (GIP) gene, or a GIP-receptor gene.

The iRNA-lipophilic conjugates described herein are not limited in thecell lines to which they can be applied or to the nucleic acids to whichthey can target.

iRNA Agent Structure

The monomers described herein can be used to make oligonucleotides whichare useful as iRNA agents, e.g., RNA molecules, (double-stranded;single-stranded) that mediate RNAi, e.g., with respect to an endogenousgene of a subject or to a gene of a pathogen. In most cases the iRNAagent will incorporate momomers described herein together with naturallyoccurring nucleosides or nucleotides or with other modified nucleosidesor nucleotides. The modified monomers can be present at any position inthe iRNA agent, e.g., at the terminii or in the middle region of an iRNAagent or in a duplex region or in an unpaired region. In a preferredembodiment iRNA agent can have any architecture, e.g., architecturedescribed herein. E.g., it can be incorporated into an iRNA agent havingan overhang structure, a hairpin or other single strand structure or atwo-strand structure, as described herein.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, ornucleoside surrogate, all of which are defined herein (see, e.g., thesection below entitled RNA Agents). While numerous modified RNAs andnucleoside surrogates are described, preferred examples include thosewhich have greater resistance to nuclease degradation than do unmodifiedRNAs.

Preferred examples include those which have a 2′ sugar modification, amodification in a single strand overhang, preferably a 3′ single strandoverhang, or, particularly if single stranded, a 5′ modification whichincludes one or more phosphate groups or one or more analogs of aphosphate group.

An “iRNA agent” as used herein, is an RNA agent which can, or which canbe cleaved into an RNA agent which can, down regulate the expression ofa target gene, preferably an endogenous or pathogen target RNA. Whilenot wishing to be bound by theory, an iRNA agent may act by one or moreof a number of mechanisms, including post-transcriptional cleavage of atarget mRNA sometimes referred to in the art as RNAi, orpre-transcriptional or pre-translational mechanisms. An iRNA agent caninclude a single strand or can include more than one strands, e.g., itcan be a double stranded iRNA agent. If the iRNA agent is a singlestrand it is particularly preferred that it include a 5′ modificationwhich includes one or more phosphate groups or one or more analogs of aphosphate group.

The RRMS-containing iRNA agent should include a region of sufficienthomology to the target gene, and be of sufficient length in terms ofnucleotides, such that the iRNA agent, or a fragment thereof, canmediate down regulation of the target gene. (For ease of exposition theterm nucleotide or ribonucleotide is sometimes used herein in referenceto one or more monomeric subunits of an RNA agent. It will be understoodherein that the usage of the term “ribonucleotide” or “nucleotide”,herein can, in the case of a modified RNA or nucleotide surrogate, alsorefer to a modified nucleotide, or surrogate replacement moiety at oneor more positions.) Thus, the iRNA agent is or includes a region whichis at least partially, and in some embodiments fully, complementary tothe target RNA. It is not necessary that there be perfectcomplementarity between the iRNA agent and the target, but thecorrespondence must be sufficient to enable the iRNA agent, or acleavage product thereof, to direct sequence specific silencing, e.g.,by RNAi cleavage of the target RNA, e.g., mRNA.

Complementarity, or degree of homology with the target strand, is mostcritical in the antisense strand. While perfect complementarity,particularly in the antisense strand, is often desired some embodimentscan include, particularly in the antisense strand, one or more butpreferably 6, 5, 4, 3, 2, or fewer mismatches (with respect to thetarget RNA). The mismatches, particularly in the antisense strand, aremost tolerated in the terminal regions and if present are preferably ina terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides ofthe 5′ and/or 3′ terminus. The sense strand need only be sufficientlycomplementary with the antisense strand to maintain the over all doublestrand character of the molecule.

As discussed elsewhere herein, an iRNA agent will often be modified orinclude nucleoside surrogates in addition to the ribose replacementmodification subunit (RRMS). Single stranded regions of an iRNA agentwill often be modified or include nucleoside surrogates, e.g., theunpaired region or regions of a hairpin structure, e.g., a region whichlinks two complementary regions, can have modifications or nucleosidesurrogates. Modification to stabilize one or more 3′- or 5′-terminus ofan iRNA agent, e.g., against exonucleases, or to favor the antisensesRNA agent to enter into RISC are also favored. Modifications caninclude C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyllinkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethyleneglycol, hexaethylene glycol), special biotin or fluorescein reagentsthat come as phosphoramidites and that have another DMT-protectedhydroxyl group, allowing multiple couplings during RNA synthesis.

iRNA agents include: molecules that are long enough to trigger theinterferon response (which can be cleaved by Dicer (Bernstein et al.2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencingcomplex)); and, molecules which are sufficiently short that they do nottrigger the interferon response (which molecules can also be cleaved byDicer and/or enter a RISC), e.g., molecules which are of a size whichallows entry into a RISC, e.g., molecules which resemble Dicer-cleavageproducts. Molecules that are short enough that they do not trigger aninterferon response are termed sRNA agents or shorter iRNA agentsherein. “sRNA agent or shorter iRNA agent” as used herein, refers to aniRNA agent, e.g., a double stranded RNA agent or single strand agent,that is sufficiently short that it does not induce a deleteriousinterferon response in a human cell, e.g., it has a duplexed region ofless than 60 but preferably less than 50, 40, or 30 nucleotide pairs.The sRNA agent, or a cleavage product thereof, can down regulate atarget gene, e.g., by inducing RNAi with respect to a target RNA,preferably an endogenous or pathogen target RNA.

Each strand of an sRNA agent can be equal to or less than 30, 25, 24,23, 22, 21, or 20 nucleotides in length. The strand is preferably atleast 19 nucleotides in length. For example, each strand can be between21 and 25 nucleotides in length. Preferred sRNA agents have a duplexregion of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, andone or more overhangs, preferably one or two 3′ overhangs, of 2-3nucleotides.

In addition to homology to target RNA and the ability to down regulate atarget gene, an iRNA agent will preferably have one or more of thefollowing properties:

(1) it will be of the Formula 1, 2, 3, or 4 set out in the RNA Agentsection below;

(2) if single stranded it will have a 5′ modification which includes oneor more phosphate groups or one or more analogs of a phosphate group;

(3) it will, despite modifications, even to a very large number, or allof the nucleosides, have an antisense strand that can present bases (ormodified bases) in the proper three dimensional framework so as to beable to form correct base pairing and form a duplex structure with ahomologous target RNA which is sufficient to allow down regulation ofthe target, e.g., by cleavage of the target RNA;

(4) it will, despite modifications, even to a very large number, or allof the nucleosides, still have “RNA-like” properties, i.e., it willpossess the overall structural, chemical and physical properties of anRNA molecule, even though not exclusively, or even partly, ofribonucleotide-based content. For example, an iRNA agent can contain,e.g., a sense and/or an antisense strand in which all of the nucleotidesugars contain e.g., 2′ fluoro in place of 2′ hydroxyl. Thisdeoxyribonucleotide-containing agent can still be expected to exhibitRNA-like properties. While not wishing to be bound by theory, theelectronegative fluorine prefers an axial orientation when attached tothe C2′ position of ribose. This spatial preference of fluorine can, inturn, force the sugars to adopt a C₃′-endo pucker. This is the samepuckering mode as observed in RNA molecules and gives rise to theRNA-characteristic A-family-type helix.

Further, since fluorine is a good hydrogen bond acceptor, it canparticipate in the same hydrogen bonding interactions with watermolecules that are known to stabilize RNA structures. (Generally, it ispreferred that a modified moiety at the 2′ sugar position will be ableto enter into H-bonding which is more characteristic of the OH moiety ofa ribonucleotide than the H moiety of a deoxyribonucleotide. A preferrediRNA agent will: exhibit a C₃′-endo pucker in all, or at least 50, 75,80, 85, 90, or 95% of its sugars; exhibit a C₃′-endo pucker in asufficient amount of its sugars that it can give rise to a theRNA-characteristic A-family-type helix; will have no more than 20, 10,5, 4, 3, 2, orl sugar which is not a C₃′-endo pucker structure. Theselimitations are particularly preferably in the antisense strand;

(5) regardless of the nature of the modification, and even though theRNA agent can contain deoxynucleotides or modified deoxynucleotides,particularly in overhang or other single strand regions, it is preferredthat DNA molecules, or any molecule in which more than 50, 60, or 70% ofthe nucleotides in the molecule, or more than 50, 60, or 70% of thenucleotides in a duplexed region are deoxyribonucleotides, or modifieddeoxyribonucleotides which are deoxy at the 2′ position, are excludedfrom the definition of RNA agent.

A “single strand iRNA agent” as used herein, is an iRNA agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpan-handle structure. Single strand iRNA agents are preferably antisensewith regard to the target molecule. In preferred embodiments singlestrand iRNA agents are 5′ phosphorylated or include a phosphoryl analogat the 5′ prime terminus 5′-phosphate modifications include those whichare compatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)₂(O)P—O-5′); 5′-diphosphate((HO)₂(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-). (These modifications can also be used with theantisense strand of a double stranded iRNA.)

A single strand iRNA agent should be sufficiently long that it can enterthe RISC and participate in RISC mediated cleavage of a target mRNA. Asingle strand iRNA agent is at least 14, and more preferably at least15, 20, 25, 29, 35, 40, or 50 nucleotides in length. It is preferablyless than 200, 100, or 60 nucleotides in length.

Hairpin iRNA agents will have a duplex region equal to or at least 17,18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex regionwill preferably be equal to or less than 200, 100, or 50, in length.Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23,and 19 to 21 nucleotides pairs in length. The hairpin will preferablyhave a single strand overhang or terminal unpaired region, preferablythe 3′, and preferably of the antisense side of the hairpin. Preferredoverhangs are 2-3 nucleotides in length.

A “double stranded (ds) iRNA agent” as used herein, is an iRNA agentwhich includes more than one, and preferably two, strands in whichinterchain hybridization can form a region of duplex structure.

The antisense strand of a double stranded iRNA agent should be equal toor at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides inlength. It should be equal to or less than 200, 100, or 50, nucleotidesin length. Preferred ranges are 17 to 25, 19 to 23, and 19 to 21nucleotides in length.

The sense strand of a double stranded iRNA agent should be equal to orat least 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length.It should be equal to or less than 200, 100, or 50, nucleotides inlength. Preferred ranges are 17 to 25, 19 to 23, and 19 to 21nucleotides in length.

The double strand portion of a double stranded iRNA agent should beequal to or at least, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29,40, or 60 nucleotide pairs in length. It should be equal to or less than200, 100, or 50, nucleotides pairs in length. Preferred ranges are15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.

In many embodiments, the ds iRNA agent is sufficiently large that it canbe cleaved by an endogenous molecule, e.g., by Dicer, to produce smallerds iRNA agents, e.g., sRNAs agents

It may be desirable to modify one or both of the antisense and sensestrands of a double strand iRNA agent. In some cases they will have thesame modification or the same class of modification but in other casesthe sense and antisense strand will have different modifications, e.g.,in some cases it is desirable to modify only the sense strand. It may bedesirable to modify only the sense strand, e.g., to inactivate it, e.g.,the sense strand can be modified in order to inactivate the sense strandand prevent formation of an active sRNA/protein or RISC. This can beaccomplished by a modification which prevents 5′-phosphorylation of thesense strand, e.g., by modification with a 5′-O-methyl ribonucleotide(see Nykänen et al., (2001) ATP requirements and small interfering RNAstructure in the RNA interference pathway. Cell 107, 309-321.)

Other modifications which prevent phosphorylation can also be used,e.g., simply substituting the 5′-OH by H rather than O-Me.Alternatively, a large bulky group may be added to the 5′-phosphateturning it into a phosphodiester linkage, though this may be lessdesirable as phosphodiesterases can cleave such a linkage and release afunctional sRNA 5′-end. Antisense strand modifications include 5′phosphorylation as well as any of the other 5′ modifications discussedherein, particularly the 5′ modifications discussed above in the sectionon single stranded iRNA molecules.

It is preferred that the sense and antisense strands be chosen such thatthe ds iRNA agent includes a single strand or unpaired region at one orboth ends of the molecule. Thus, a ds iRNA agent contains sense andantisense strands, preferable paired to contain an overhang, e.g., oneor two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3nucleotides. Most embodiments will have a 3′ overhang. Preferred sRNAagents will have single-stranded overhangs, preferably 3′ overhangs, of1 or preferably 2 or 3 nucleotides in length at each end. The overhangscan be the result of one strand being longer than the other, or theresult of two strands of the same length being staggered. 5′ ends arepreferably phosphorylated.

Preferred lengths for the duplexed region is between 15 and 30, mostpreferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe sRNA agent range discussed above. sRNA agents can resemble in lengthand structure the natural Dicer processed products from long dsRNAs.Embodiments in which the two strands of the sRNA agent are linked, e.g.,covalently linked are also included. Hairpin, or other single strandstructures which provide the required double stranded region, andpreferably a 3′ overhang are also within the invention.

The isolated iRNA agents described herein, including ds iRNA agents andsRNA agents can mediate silencing of a target RNA, e.g., mRNA, e.g., atranscript of a gene that encodes a protein. For convenience, such mRNAis also referred to herein as mRNA to be silenced. Such a gene is alsoreferred to as a target gene. In general, the RNA to be silenced is anendogenous gene or a pathogen gene. In addition, RNAs other than mRNA,e.g., tRNAs, and viral RNAs, can also be targeted.

As used herein, the phrase “mediates RNAi” refers to the ability tosilence, in a sequence specific manner, a target RNA. While not wishingto be bound by theory, it is believed that silencing uses the RNAimachinery or process and a guide RNA, e.g., an sRNA agent of 21 to 23nucleotides.

As used herein, “specifically hybridizable” and “complementary” areterms which are used to indicate a sufficient degree of complementaritysuch that stable and specific binding occurs between a compound of theinvention and a target RNA molecule. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.The non-target sequences typically differ by at least 5 nucleotides.

In one embodiment, an iRNA agent is “sufficiently complementary” to atarget RNA, e.g., a target mRNA, such that the iRNA agent silencesproduction of protein encoded by the target mRNA. In another embodiment,the iRNA agent is “exactly complementary” (excluding the RRMS containingsubunit(s)) to a target RNA, e.g., the target RNA and the iRNA agentanneal, preferably to form a hybrid made exclusively of Watson-Crickbasepairs in the region of exact complementarity. A “sufficientlycomplementary” target RNA can include an internal region (e.g., of atleast 10 nucleotides) that is exactly complementary to a target RNA.Moreover, in some embodiments, the iRNA agent specifically discriminatesa single-nucleotide difference. In this case, the iRNA agent onlymediates RNAi if exact complementary is found in the region (e.g.,within 7 nucleotides of) the single-nucleotide difference.

As used herein, the term “oligonucleotide” refers to a nucleic acidmolecule (RNA or DNA) preferably of length less than 100, 200, 300, or400 nucleotides.

RNA agents discussed herein include otherwise unmodified RNA as well asRNA which have been modified, e.g., to improve efficacy, and polymers ofnucleoside surrogates. Unmodified RNA refers to a molecule in which thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are the same or essentially the same as that which occur innature, preferably as occur naturally in the human body. The art hasreferred to rare or unusual, but naturally occurring, RNAs as modifiedRNAs, see, e.g., Limbach et al., (1994) Summary: the modifiednucleosides of RNA, Nucleic Acids Res. 22: 2183-2196. Such rare orunusual RNAs, often termed modified RNAs (apparently because the aretypically the result of a post transcriptionally modification) arewithin the term unmodified RNA, as used herein. Modified RNA as usedherein refers to a molecule in which one or more of the components ofthe nucleic acid, namely sugars, bases, and phosphate moieties, aredifferent from that which occur in nature, preferably different fromthat which occurs in the human body. While they are referred to asmodified “RNAs,” they will of course, because of the modification,include molecules which are not RNAs. Nucleoside surrogates aremolecules in which the ribophosphate backbone is replaced with anon-ribophosphate construct that allows the bases to the presented inthe correct spatial relationship such that hybridization issubstantially similar to what is seen with a ribophosphate backbone,e.g., non-charged mimics of the ribophosphate backbone. Examples of allof the above are discussed herein.

Much of the discussion below refers to single strand molecules. In manyembodiments of the invention a double stranded iRNA agent, e.g., apartially double stranded iRNA agent, is required or preferred. Thus, itis understood that that double stranded structures (e.g. where twoseparate molecules are contacted to form the double stranded region orwhere the double stranded region is formed by intramolecular pairing(e.g., a hairpin structure)) made of the single stranded structuresdescribed below are within the invention. Preferred lengths aredescribed elsewhere herein.

As nucleic acids are polymers of subunits or monomers, many of themodifications described below occur at a position which is repeatedwithin a nucleic acid, e.g., a modification of a base, or a phosphatemoiety, or the a non-linking O of a phosphate moiety. In some cases themodification will occur at all of the subject positions in the nucleicacid but in many, and infact in most cases it will not. By way ofexample, a modification may only occur at a 3′ or 5′ terminal position,may only occur in a terminal regions, e.g. at a position on a terminalnucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. Amodification may occur in a double strand region, a single strandregion, or in both. A modification may occur only in the double strandregion of an RNA or may only occur in a single strand region of an RNA.E.g., a phosphorothioate modification at a non-linking O position mayonly occur at one or both termini, may only occur in a terminal regions,e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5,or 10 nucleotides of a strand, or may occur in double strand and singlestrand regions, particularly at termini. The 5′ end or ends can bephosphorylated.

In some embodiments it is particularly preferred, e.g., to enhancestability, to include particular bases in overhangs, or to includemodified nucleotides or nucleotide surrogates, in single strandoverhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can bedesirable to include purine nucleotides in overhangs. In someembodiments all or some of the bases in a 3′ or 5′ overhang will bemodified, e.g., with a modification described herein. Modifications caninclude, e.g., the use of modifications at the 2′ OH group of the ribosesugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine,instead of ribonucleotides, and modifications in the phosphate group,e.g., phosphothioate modifications. Overhangs need not be homologouswith the target sequence.

Modifications and nucleotide surrogates are discussed below.

The scaffold presented above in Formula 1 represents a portion of aribonucleic acid. The basic components are the ribose sugar, the base,the terminal phosphates, and phosphate internucleotide linkers. Wherethe bases are naturally occurring bases, e.g., adenine, uracil, guanineor cytosine, the sugars are the unmodified 2′ hydroxyl ribose sugar (asdepicted) and W, X, Y, and Z are all O, Formula 1 represents a naturallyoccurring unmodified oligoribonucleotide.

Unmodified oligoribonucleotides may be less than optimal in someapplications, e.g., unmodified oligoribonucleotides can be prone todegradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleicacid phosphodiester bonds. However, chemical modifications to one ormore of the above RNA components can confer improved properties, and,e.g., can render oligoribonucleotides more stable to nucleases.Umodified oligoribonucleotides may also be less than optimal in terms ofoffering tethering points for attaching ligands or other moieties to aniRNA agent.

Modified nucleic acids and nucleotide surrogates can include one or moreof:

(i) alteration, e.g., replacement, of one or both of the non-linking (Xand Y) phosphate oxygens and/or of one or more of the linking (W and Z)phosphate oxygens (When the phosphate is in the terminal position, oneof the positions W or Z will not link the phosphate to an additionalelement in a naturally occurring ribonucleic acid. However, forsimplicity of terminology, except where otherwise noted, the W positionat the 5′ end of a nucleic acid and the terminal Z position at the 3′end of a nucleic acid, are within the term “linking phosphate oxygens”as used herein.);

(ii) alteration, e.g., replacement, of a constituent of the ribosesugar, e.g., of the 2′ hydroxyl on the ribose sugar, or wholesalereplacement of the ribose sugar with a structure other than ribose,e.g., as described herein;

(iii) wholesale replacement of the phosphate moiety (bracket I) with“dephospho” linkers;

(iv) modification or replacement of a naturally occurring base;

(v) replacement or modification of the ribose-phosphate backbone(bracket II);

(vi) modification of the 3′ end or 5′ end of the RNA, e.g., removal,modification or replacement of a terminal phosphate group or conjugationof a moiety, e.g. a fluorescently labeled moiety, to either the 3′ or 5′end of RNA.

The terms replacement, modification, alteration, and the like, as usedin this context, do not imply any process limitation, e.g., modificationdoes not mean that one must start with a reference or naturallyoccurring ribonucleic acid and modify it to produce a modifiedribonucleic acid bur rather modified simply indicates a difference froma naturally occurring molecule.

It is understood that the actual electronic structure of some chemicalentities cannot be adequately represented by only one canonical form(i.e. Lewis structure). While not wishing to be bound by theory, theactual structure can instead be some hybrid or weighted average of twoor more canonical forms, known collectively as resonance forms orstructures. Resonance structures are not discrete chemical entities andexist only on paper. They differ from one another only in the placementor “localization” of the bonding and nonbonding electrons for aparticular chemical entity. It can be possible for one resonancestructure to contribute to a greater extent to the hybrid than theothers. Thus, the written and graphical descriptions of the embodimentsof the present invention are made in terms of what the art recognizes asthe predominant resonance form for a particular species. For example,any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen)would be represented by X═O and Y═N in the above figure.

Specific modifications are discussed in more detail below.

The Phosphate Group

The phosphate group is a negatively charged species. The charge isdistributed equally over the two non-linking oxygen atoms (i.e., X and Yin Formula 1 above). However, the phosphate group can be modified byreplacing one of the oxygens with a different substituent. One result ofthis modification to RNA phosphate backbones can be increased resistanceof the oligoribonucleotide to nucleolytic breakdown. Thus while notwishing to be bound by theory, it can be desirable in some embodimentsto introduce alterations which result in either an uncharged linker or acharged linker with unsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. Unlike the situation where only one of X or Y isaltered, the phosphorus center in the phosphorodithioates is achiralwhich precludes the formation of oligoribonucleotides diastereomers.Diastereomer formation can result in a preparation in which theindividual diastereomers exhibit varying resistance to nucleases.Further, the hybridization affinity of RNA containing chiral phosphategroups can be lower relative to the corresponding unmodified RNAspecies. Thus, while not wishing to be bound by theory, modifications toboth X and Y which eliminate the chiral center, e.g. phosphorodithioateformation, may be desirable in that they cannot produce diastereomermixtures. Thus, X can be any one of S, Se, B, C, H, N, or OR(R is alkylor aryl). Thus Y can be any one of S, Se, B, C, H, N, or OR(R is alkylor aryl). Replacement of X and/or Y with sulfur is preferred.

The phosphate linker can also be modified by replacement of a linkingoxygen (i.e., W or Z in Formula 1) with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylenephosphonates). The replacement can occur at a terminaloxygen (position W (3′) or position Z (5′). Replacement of W with carbonor Z with nitrogen is preferred.

Candidate agents can be evaluated for suitability as described below.

The Sugar Group

A modified RNA can include modification of all or some of the sugargroups of the ribonucleic acid. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents. While not being bound by theory, enhanced stability isexpected since the hydroxyl can no longer be deprotonated to form a 2′alkoxide ion. The 2′ alkoxide can catalyze degradation by intramolecularnucleophilic attack on the linker phosphorus atom. Again, while notwishing to be bound by theory, it can be desirable to some embodimentsto introduce alterations in which alkoxide formation at the 2′ positionis not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE(AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino) Itis noteworthy that oligonucleotides containing only the methoxyethylgroup (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nucleasestabilities comparable to those modified with the robustphosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino), —NHC(O)R(R=alkyl, cycloalkyl,aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino functionality. Preferredsubstitutents are 2′-methoxyethyl, 2′-OCH₃,2′-O-allyl, 2′-C— allyl, and2′-fluoro.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified RNA can include nucleotidescontaining e.g., arabinose, as the sugar.

Modified RNAs can also include “abasic” sugars, which lack a nucleobaseat C-1′. These abasic sugars can also be further contain modificationsat one or more of the constituent sugar atoms.

To maximize nuclease resistance, the 2′ modifications can be used incombination with one or more phosphate linker modifications (e.g.,phosphorothioate). The so-called “chimeric” oligonucleotides are thosethat contain two or more different modifications.

The modificaton can also entail the wholesale replacement of a ribosestructure with another entity at one or more sites in the iRNA agent.These modifications are described in section entitled RiboseReplacements for RRMSs.

Candidate modifications can be evaluated as described below.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containingconnectors (cf.

Bracket I in Formula 1 above). While not wishing to be bound by theory,it is believed that since the charged phosphodiester group is thereaction center in nucleolytic degradation, its replacement with neutralstructural mimics should impart enhanced nuclease stability. Again,while not wishing to be bound by theory, it can be desirable, in someembodiment, to introduce alterations in which the charged phosphategroup is replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group includesiloxane, carbonate, carboxymethyl, carbamate, amide, thioether,ethylene oxide linker, sulfonate, sulfonamide, thioformacetal,formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.Preferred replacements include the methylenecarbonylamino andmethylenemethylimino groups.

Candidate modifications can be evaluated as described below.

Replacement of Ribophosphate Backbone

Oligonucleotide—mimicking scaffolds can also be constructed wherein thephosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates (see Bracket II of Formula 1 above).While not wishing to be bound by theory, it is believed that the absenceof a repetitively charged backbone diminishes binding to proteins thatrecognize polyanions (e.g. nucleases). Again, while not wishing to bebound by theory, it can be desirable in some embodiment, to introducealterations in which the bases are tethered by a neutral surrogatebackbone.

Examples include the mophilino, cyclobutyl, pyrrolidine and peptidenucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNAsurrogate. Candidate modifications can be evaluated as described below.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.They can include modification or replacement of an entire terminalphosphate or of one or more of the atoms of the phosphate group. E.g.,the 3′ and 5′ ends of an oligonucleotide can be conjugated to otherfunctional molecular entities such as labeling moieties, e.g.,fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) orprotecting groups (based e.g., on sulfur, silicon, boron or ester). Thefunctional molecular entities can be attached to the sugar through aphosphate group and/or a spacer. The terminal atom of the spacer canconnect to or replace the linking atom of the phosphate group or theC-3′ or C-5′ 0, N, S or C group of the sugar. Alternatively, the spacercan connect to or replace the terminal atom of a nucleotide surrogate(e.g., PNAs). These spacers or linkers can include e.g., —(CH₂)_(n)—,—(CH₂)_(n)N—, —(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g.,n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine,thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotinand fluorescein reagents. When a spacer/phosphate-functional molecularentity-spacer/phosphate array is interposed between two strands of iRNAagents, this array can substitute for a hairpin RNA loop in ahairpin-type RNA agent. The 3′ end can be an —OH group. While notwishing to be bound by theory, it is believed that conjugation ofcertain moieties can improve transport, hybridization, and specificityproperties. Again, while not wishing to be bound by theory, it may bedesirable to introduce terminal alterations that improve nucleaseresistance. Other examples of terminal modifications include dyes,intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene,mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclicaromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol,cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,bisimidazole, histamine, imidazole clusters, acridine-imidazoleconjugates, Eu3+ complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, includingas discussed elsewhere herein to modulate activity or to modulateresistance to degradation. Terminal modifications useful for modulatingactivity include modification of the 5′ end with phosphate or phosphateanalogs. E.g., in preferred embodiments iRNA agents, especiallyantisense strands, are 5′ phosphorylated or include a phosphoryl analogat the 5′ prime terminus. 5′-phosphate modifications include those whichare compatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)₂(O)P—O-5′); 5′-diphosphate((HO)₂(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH₂—), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).

Terminal modifications can also be useful for monitoring distribution,and in such cases the preferred groups to be added include fluorophores,e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminalmodifications can also be useful for enhancing uptake, usefulmodifications for this include cholesterol. Terminal modifications canalso be useful for cross-linking an RNA agent to another moiety;modifications useful for this include mitomycin C.

Candidate modifications can be evaluated as described below.

The Bases

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. E.g., nuclease resistant oligoribonucleotides canbe prepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogs of any of the abovebases, e.g., “unusual bases” and “universal bases” described herein, canbe employed. Examples include without limitation 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2,N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine,7-deazaadenine, N6, N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N-6-isopentenyladenine, N-methylguanines, or O-alkylatedbases.

Further purines and pyrimidines include those disclosed in U.S. Pat. No.3,687,808, those disclosed in the Concise Encyclopedia Of PolymerScience And Engineering, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, and those disclosed by Englisch et al., AngewandteChemie, International Edition, 1991, 30, 613.

Generally, base changes are less preferred for promoting stability, butthey can be useful for other reasons, e.g., some, e.g.,2,6-diaminopurine and 2 amino purine, are fluorescent.

Modified bases can reduce target specificity. This should be taken intoconsideration in the design of iRNA agents.

Candidate modifications can be evaluated as described below.

Evaluation of Candidate RNA's

One can evaluate a candidate RNA agent, e.g., a modified RNA, for aselected property by exposing the agent or modified molecule and acontrol molecule to the appropriate conditions and evaluating for thepresence of the selected property. For example, resistance to adegradent can be evaluated as follows. A candidate modified RNA (andpreferably a control molecule, usually the unmodified form) can beexposed to degradative conditions, e.g., exposed to a milieu, whichincludes a degradative agent, e.g., a nuclease. E.g., one can use abiological sample, e.g., one that is similar to a milieu, which might beencountered, in therapeutic use, e.g., blood or a cellular fraction,e.g., a cell-free homogenate or disrupted cells. The candidate andcontrol could then be evaluated for resistance to degradation by any ofa number of approaches. For example, the candidate and control could belabeled, preferably prior to exposure, with, e.g., a radioactive orenzymatic label, or a fluorescent label, such as Cy3 or Cy5. Control andmodified RNA's can be incubated with the degradative agent, andoptionally a control, e.g., an inactivated, e.g., heat inactivated,degradative agent. A physical parameter, e.g., size, of the modified andcontrol molecules are then determined. They can be determined by aphysical method, e.g., by polyacrylamide gel electrophoresis or a sizingcolumn, to assess whether the molecule has maintained its originallength, or assessed functionally. Alternatively, Northern blot analysiscan be used to assay the length of an unlabeled modified molecule.

A functional assay can also be used to evaluate the candidate agent. Afunctional assay can be applied initially or after an earliernon-functional assay, (e.g., assay for resistance to degradation) todetermine if the modification alters the ability of the molecule tosilence gene expression. For example, a cell, e.g., a mammalian cell,such as a mouse or human cell, can be co-transfected with a plasmidexpressing a fluorescent protein, e.g., GFP, and a candidate RNA agenthomologous to the transcript encoding the fluorescent protein (see,e.g., WO 00/44914). For example, a modified dsRNA homologous to the GFPmRNA can be assayed for the ability to inhibit GFP expression bymonitoring for a decrease in cell fluorescence, as compared to a controlcell, in which the transfection did not include the candidate dsRNA,e.g., controls with no agent added and/or controls with a non-modifiedRNA added. Efficacy of the candidate agent on gene expression can beassessed by comparing cell fluorescence in the presence of the modifiedand unmodified dsRNA agents.

In an alternative functional assay, a candidate dsRNA agent homologousto an endogenous mouse gene, preferably a maternally expressed gene,such as c-mos, can be injected into an immature mouse oocyte to assessthe ability of the agent to inhibit gene expression in vivo (see, e.g.,WO 01/36646). A phenotype of the oocyte, e.g., the ability to maintainarrest in metaphase II, can be monitored as an indicator that the agentis inhibiting expression. For example, cleavage of c-mos mRNA by a dsRNAagent would cause the oocyte to exit metaphase arrest and initiateparthenogenetic development (Colledge et al. Nature 370: 65-68, 1994;Hashimoto et al. Nature, 370:68-71, 1994). The effect of the modifiedagent on target RNA levels can be verified by Northern blot to assay fora decrease in the level of target mRNA, or by Western blot to assay fora decrease in the level of target protein, as compared to a negativecontrol. Controls can include cells in which with no agent is addedand/or cells in which a non-modified RNA is added.

REFERENCES General References

The oligoribonucleotides and oligoribonucleosides used in accordancewith this invention may be with solid phase synthesis, see for example“Oligonucleotide synthesis, a practical approach”, Ed. M. J. Gait, IRLPress, 1984; “Oligonucleotides and Analogues, A Practical Approach”, Ed.F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine-aidedmethods of oligodeoxyribonucleotide synthesis, Chapter 2,Oligoribonucleotide synthesis, Chapter3,2′-O-Methyloligoribonucleotide—s: synthesis and applications, Chapter4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis ofoligonucleotide phosphorodithioates, Chapter 6, Synthesis ofoligo-2′-deoxyribonucleoside methylphosphonates, and Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Hely. Chian. Acta, 1995, 78,486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein.

Modification described in WO 00/44895, WO01/75164, or WO02/44321 can beused herein.

The disclosure of all publications, patents, and published patentapplications listed herein are hereby incorporated by reference.

Phosphate Group References

The preparation of phosphinate oligoribonucleotides is described in U.S.Pat. No. 5,508,270. The preparation of alkyl phosphonateoligoribonucleotides is described in U.S. Pat. No. 4,469,863. Thepreparation of phosphoramidite oligoribonucleotides is described in U.S.Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation ofphosphotriester oligoribonucleotides is described in U.S. Pat. No.5,023,243. The preparation of borano phosphate oligoribonucleotide isdescribed in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of3′-Deoxy-3′-amino phosphoramidate oligoribonucleotides is described inU.S. Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonateoligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001,66, 2789-2801. Preparation of sulfur bridged nucleotides is described inSproat et al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al.Tetrahedron Lett. 1989, 30, 4693.

Sugar Group References

Modifications to the 2′ modifications can be found in Verma, S. et al.Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein.Specific modifications to the ribose can be found in the followingreferences: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36,831-841), 2′-MOE (Martin, P. Hely. Chim. Acta 1996, 79, 1930-1938),“LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Replacement of the Phosphate Group References

Methylenemethylimino linked oligoribonucleosides, also identified hereinas MMI linked oligoribonucleosides, methylenedimethylhydrazo linkedoligoribonucleosides, also identified herein as MDH linkedoligoribonucleosides, and methylenecarbonylamino linkedoligonucleosides, also identified herein as amide-3 linkedoligoribonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified herein as amide-4 linkedoligoribonucleosides as well as mixed backbone compounds having, as forinstance, alternating MMI and PO or PS linkages can be prepared as isdescribed in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and inpublished PCT applications PCT/US92/04294 and PCT/US92/04305 (publishedas WO 92/20822 WO and 92/20823, respectively). Formacetal andthioformacetal linked oligoribonucleosides can be prepared as isdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxidelinked oligoribonucleosides can be prepared as is described in U.S. Pat.No. 5,223,618. Siloxane replacements are described in Cormier, J. F. etal. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements aredescribed in Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethylreplacements are described in Edge, M. D. et al. J. Chem. Soc. PerkinTrans. 11972, 1991. Carbamate replacements are described in Stirchak, E.P. Nucleic Acids Res. 1989, 17, 6129.

Replacement of the Phosphate-Ribose Backbone References

Cyclobutyl sugar surrogate compounds can be prepared as is described inU.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared asis described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates canbe prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033,and other related patent disclosures. Peptide Nucleic Acids (PNAs) areknown per se and can be prepared in accordance with any of the variousprocedures referred to in Peptide Nucleic Acids (PNA): Synthesis,Properties and Potential Applications, Bioorganic & Medicinal Chemistry,1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat.No. 5,539,083.

Terminal Modification References

Terminal modifications are described in Manoharan, M. et al. Antisenseand Nucleic Acid Drug Development 12, 103-128 (2002) and referencestherein.

Bases References

N-2 substitued purine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amiditescan be prepared as is described in U.S. Pat. No. 5,457,191.5,6-Substituted pyrimidine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleosideamidites can be prepared as is described in U.S. Pat. No. 5,484,908.Additional references can be disclosed in the above section on basemodifications.

Preferred iRNA Agents

Preferred RNA agents have the following structure (see Formula 2 below):

Referring to Formula 2 above, R¹, R², and R³ are each, independently, H,(i.e. abasic nucleotides), adenine, guanine, cytosine and uracil,inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine,isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine,6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other8-substituted adenines and guanines, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine, 5-substitutedpyrimidines, 6-azapyrimidines and N2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil,N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone,5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil,3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine,N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N-6-isopentenyladenine,N-methylguanines, or O-alkylated bases.

R⁴, R⁵, and R⁶ are each, independently, OR⁸, O(CH₂CH₂O)_(n)CH₂CH₂OR⁸;O(CH₂)_(n)R⁹; O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂;NH(CH₂CH₂NH)_(n)CH₂CH₂NHR⁹; NHC(O)R⁸; cyano; mercapto, SR⁸;alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl,alkynyl, each of which may be optionally substituted with halo, hydroxy,oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy,amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,alkylcarbonyl, acyloxy, cyano, or ureido; or R⁴, R⁵, or R⁶ togethercombine with R⁷ to form an [-O—CH₂—] covalently bound bridge between thesugar 2′ and 4′ carbons.

A¹ is:

H; OH; OCH₃; W¹; an abasic nucleotide; or absent;

(a preferred Al, especially with regard to anti-sense strands, is chosenfrom 5′-monophosphate ((HO)₂(O)P—O-5′), 5′-diphosphate((HO)₂(O)P—O—P(HO)(O)—O-5′), 5′-triphosphate((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′),5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P-β-(HO)(O)P—O—P(HO)(O)—O-5′),5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′),5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH₂—), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-)).

A² is:

A³ is:

and

A⁴ is:

H; Z⁴; an inverted nucleotide; an abasic nucleotide; or absent.

W¹ is OH, (CH₂)_(n)R¹⁰, (CH₂)_(n)NHR¹⁰, O(CH₂)_(n) OR^(b), (CH₂)_(n)SR¹⁰; O(CH₂)_(n)R¹⁰; O(CH₂)_(n)OR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)SR¹⁰;O(CH₂)_(n)SS(CH₂)_(n)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰, NH(CH₂)_(n)R¹⁰;NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰,S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)OR¹⁰, S(CH₂)_(n)SR¹⁰ O(CH₂CH₂O)_(n)CH₂CH₂OR¹⁰;O(CH₂CH₂O)_(m)CH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰N-Q-R¹⁰, S-Q-R¹⁰ or —O—. W⁴ is O, CH₂, NH, or S.

X′, X², X³, and X⁴ are each, independently, O or S.

Y¹, Y², Y³, and Y⁴ are each, independently, OH, O⁻, OR⁸, S, Se, BH₃ ⁻,H, NHR⁹, N(R⁹)₂ alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each ofwhich may be optionally substituted.

Z¹, Z², and Z³ are each independently O, CH₂, NH, or S. Z⁴ is OH,(CH₂)_(n)R¹⁰, (CH₂)_(n)NHR¹⁰, (CH₂)_(n) OR¹⁰, (CH₂)_(n) SR¹⁰;O(CH₂)_(n)R¹⁰; O(CH₂)_(n)OR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)SR¹⁰,O(CH₂)_(n)SS(CH₂)_(n)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰; NH(CH₂)_(n)R¹⁰;NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰,S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)OR¹⁰, S(CH₂)_(n)SR¹⁰ O(CH₂CH₂O)_(m)CH₂CH₂OR¹⁰,O(CH₂CH₂O)_(m)CH₂CH₂NHR¹⁰. NH(CH₂CH₂NM_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰N-Q-R¹⁰, S-Q-R¹⁰.

x is 5-100, chosen to comply with a length for an RNA agent describedherein.

R⁷ is H; or is together combined with R⁴, R⁵, or R⁶ to form an [-O—CH₂—]covalently bound bridge between the sugar 2′ and 4′ carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, aminoacid, or sugar; R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or aminoacid; and R¹⁰ is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5dyes); sulfur, silicon, boron or ester protecting group; intercalatingagents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatichydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), lipohilic carriers (cholesterol, cholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmiticacid,myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenicacid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino; alkyl,cycloalkyl, aryl, aralkyl, heteroaryl; radiolabelled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,bisimidazole, histamine, imidazole clusters, acridine-imidazoleconjugates, Eu3+ complexes of tetraazamacrocycles); or an RNA agent. mis O-1,000,000, and n is O-20. Q is a spacer selected from the groupconsisting of abasic sugar, amide, carboxy, oxyamine, oxyimine,thioether, disulfide, thiourea, sulfonamide, or morpholino, biotin orfluorescein reagents.

Preferred RNA agents in which the entire phosphate group has beenreplaced have the following structure (see Formula 3 below):

Referring to Formula 3, A¹⁰-A⁴⁰ is L-G-L; A¹⁰ and/or A⁴⁰ may be absent,in which L is a linker, wherein one or both L may be present or absentand is selected from the group consisting of CH₂(CH₂)_(g); N(CH₂)_(g);O(CH₂)_(g); S(CH₂)_(g). G is a functional group selected from the groupconsisting of siloxane, carbonate, carboxymethyl, carbamate, amide,thioether, ethylene oxide linker, sulfonate, sulfonamide,thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

R¹⁰, R²⁰, and R³⁰ are each, independently, H, (i.e. abasic nucleotides),adenine, guanine, cytosine and uracil, inosine, thymine, xanthine,hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N2, N-6and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil,N3-methyluracil substituted 1,2,4-triazoles, 2-pyridinone,5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil,3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine,N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N-6-isopentenyladenine,N-methylguanines, or O-alkylated bases.

R⁴⁰, R⁵⁰, and R⁶⁰ are each, independently, OR⁸, O(CH₂CH₂O)_(n)CH₂CH₂OR⁸;O(CH₂)_(n)R⁹; O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂;NH(CH₂CH₂NH)_(n)CH₂CH₂R⁹; NHC(O)R⁸;; cyano; mercapto, SR⁷;alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl,alkynyl, each of which may be optionally substituted with halo, hydroxy,oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy,amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,alkylcarbonyl, acyloxy, cyano, and ureido groups; or R⁴⁰, R⁵⁰, or R⁶⁰together combine with R⁷⁰ to form an [—O—CH₂—] covalently bound bridgebetween the sugar 2′ and 4′ carbons.

x is 5-100 or chosen to comply with a length for an RNA agent describedherein.

R⁷⁰ is H; or is together combined with R⁴⁰, R⁵⁰, or R⁶⁰ to form an[—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, aminoacid, or sugar; and R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or aminoacid. m is O-1,000,000, n is O-20, and g is O-2.

Preferred nucleoside surrogates have the following structure (seeFormula 4 below):

SLR¹⁰⁰-(M—SLR²⁰⁰)_(x)-M—SLR³⁰⁰  FORMULA 4

S is a nucleoside surrogate selected from the group consisting ofmophilino, cyclobutyl, pyrrolidine and peptide nucleic acid. L is alinker and is selected from the group consisting of CH₂(CH₂)_(g);N(CH₂)_(g); O(CH₂)_(g); S(CH₂)_(g); —C(O)(CH₂)_(n)— or may be absent. Mis an amide bond; sulfonamide; sulfinate; phosphate group; modifiedphosphate group as described herein; or may be absent.

R¹⁰⁰, R²⁰⁰, and R³⁰⁰ are each, independently, H (i.e., abasicnucleotides), adenine, guanine, cytosine and uracil, inosine, thymine,xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo,amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines andguanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidinesand N2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1,2, 4,-triazoles, 2-pyridinones, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N-6-isopentenyladenine, N-methylguanines, or O-alkylatedbases.

x is 5-100, or chosen to comply with a length for an RNA agent describedherein; and g is 0-2.

Nuclease Resistant Monomers

The monomers and methods described herein can be used to prepare an RNA,e.g., an iRNA agent, that incorporates a nuclease resistant monomer(NRM), such as those described herein and those described in copending,co-owned U.S. Provisional Application Ser. No. 60/469,612, filed on May9, 2003, and International Application No. PCT/US04/07070, both of whichare hereby incorporated by reference.

An iRNA agent can include monomers which have been modified so as toinhibit degradation, e.g., by nucleases, e.g., endonucleases orexonucleases, found in the body of a subject. These monomers arereferred to herein as NRMs, or nuclease resistance promoting monomers ormodifications. In many cases these modifications will modulate otherproperties of the iRNA agent as well, e.g., the ability to interact witha protein, e.g., a transport protein, e.g., serum albumin, or a memberof the RISC(RNA-induced Silencing Complex), or the ability of the firstand second sequences to form a duplex with one another or to form aduplex with another sequence, e.g., a target molecule.

While not wishing to be bound by theory, it is believed thatmodifications of the sugar, base, and/or phosphate backbone in an iRNAagent can enhance endonuclease and exonuclease resistance, and canenhance interactions with transporter proteins and one or more of thefunctional components of the RISC complex. Preferred modifications arethose that increase exonuclease and endonuclease resistance and thusprolong the half-life of the iRNA agent prior to interaction with theRISC complex, but at the same time do not render the iRNA agentresistant to endonuclease activity in the RISC complex. Again, while notwishing to be bound by any theory, it is believed that placement of themodifications at or near the 3′ and/or 5′ end of antisense strands canresult in iRNA agents that meet the preferred nuclease resistancecriteria delineated above. Again, still while not wishing to be bound byany theory, it is believed that placement of the modifications at e.g.,the middle of a sense strand can result in iRNA agents that arerelatively less likely to undergo off-targeting.

Modifications described herein can be incorporated into anydouble-stranded RNA and RNA-like molecule described herein, e.g., aniRNA agent. An iRNA agent may include a duplex comprising a hybridizedsense and antisense strand, in which the antisense strand and/or thesense strand may include one or more of the modifications describedherein. The anti sense strand may include modifications at the 3′ endand/or the 5′ end and/or at one or more positions that occur 1-6 (e.g.,1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. The sensestrand may include modifications at the 3′ end and/or the 5′ end and/orat any one of the intervening positions between the two ends of thestrand. The iRNA agent may also include a duplex comprising twohybridized antisense strands. The first and/or the second antisensestrand may include one or more of the modifications described herein.Thus, one and/or both antisense strands may include modifications at the3′ end and/or the 5′ end and/or at one or more positions that occur 1-6(e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand.Particular configurations are discussed below.

Modifications that can be useful for producing iRNA agents that meet thepreferred nuclease resistance criteria delineated above can include oneor more of the following chemical and/or stereochemical modifications ofthe sugar, base, and/or phosphate backbone:

(i) chiral (Sp) thioates. Thus, preferred NRMs include nucleotide dimerswith an enriched or pure for a particular chiral form of a modifiedphosphate group containing a heteroatom at the nonbridging position,e.g., Sp or Rp, at the position X, where this is the position normallyoccupied by the oxygen. The atom at X can also be S, Se, Nr₂, or Br_(a).When X is S, enriched or chirally pure S_(P) linkage is preferred.Enriched means at least 70, 80, 90, 95, or 99% of the preferred form.Such NRMs are discussed in more detail below;

(ii) attachment of one or more cationic groups to the sugar, base,and/or the phosphorus atom of a phosphate or modified phosphate backbonemoiety. Thus, preferred NRMs include monomers at the terminal positionderivatized at a cationic group. As the 5′ end of an antisense sequenceshould have a terminal —OH or phosphate group this NRM is preferably notused at the 5′ end of an anti-sense sequence. The group should beattached at a position on the base which minimizes interference with Hbond formation and hybridization, e.g., away form the face whichinteracts with the complementary base on the other strand, e.g, at the5′ position of a pyrimidine or a 7-position of a purine. These arediscussed in more detail below;

(iii) nonphosphate linkages at the termini. Thus, preferred NRMs includeNon-phosphate linkages, e.g., a linkage of 4 atoms which confers greaterresistance to cleavage than does a phosphate bond. Examples include 3′CH₂—NCH₃—O—CH₂-5′ and 3′ CH₂—NH—(O═)—CH₂-5′;

(iv) 3′-bridging thiophosphates and 5′-bridging thiophosphates. Thus,preferred NRM's can included these structures;

(v) L-RNA, 2′-5′ linkages, inverted linkages, α-nucleosides. Thus, otherpreferred NRM's include: L nucleosides and dimeric nucleotides derivedfrom L-nucleosides; 2′-5′ phosphate, non-phosphate and modifiedphosphate linkages (e.g., thiophosphates, phosphoramidates andboronophosphates); dimers having inverted linkages, e.g., 3′-3′ or 5′-5′linkages; monomers having an alpha linkage at the 1′ site on the sugar,e.g., the structures described herein having an alpha linkage;

(vi) conjugate groups. Thus, preferred NRM's can include e.g., atargeting moiety or a conjugated ligand described herein conjugated withthe monomer, e.g., through the sugar, base, or backbone;

(vi) abasic linkages. Thus, preferred NRM's can include an abasicmonomer, e.g., an abasic monomer as described herein (e.g., anucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclicaromatic monomer as described herein.; and

(vii) 5′-phosphonates and 5′-phosphate prodrugs. Thus, preferred NRM'sinclude monomers, preferably at the terminal position, e.g., the 5′position, in which one or more atoms of the phosphate group isderivatized with a protecting group, which protecting group or groups,are removed as a result of the action of a component in the subject'sbody, e.g, a carboxyesterase or an enzyme present in the subject's body.E.g., a phosphate prodrug in which a carboxy esterase cleaves theprotected molecule resulting in the production of a thioate anion whichattacks a carbon adjacent to the O of a phosphate and resulting in theproduction of an unprotected phosphate.

One or more different NRM modifications can be introduced into an iRNAagent or into a sequence of an iRNA agent. An NRM modification can beused more than once in a sequence or in an iRNA agent. As some NRM'sinterfere with hybridization the total number incorporated, should besuch that acceptable levels of iRNA agent duplex formation aremaintained.

In some embodiments NRM modifications are introduced into the terminalthe cleavage site or in the cleavage region of a sequence (a sensestrand or sequence) which does not target a desired sequence or gene inthe subject. This can reduce off-target silencing.

Chiral S_(P) Thioates

A modification can include the alteration, e.g., replacement, of one orboth of the non-linking (X and Y) phosphate oxygens and/or of one ormore of the linking (W and Z) phosphate oxygens. Formula X below depictsa phosphate moiety linking two sugar/sugar surrogate-base moieties, SB₁and SB₂.

In certain embodiments, one of the non-linking phosphate oxygens in thephosphate backbone moiety (X and Y) can be replaced by any one of thefollowing: S, Se, BR₃ (R is hydrogen, alkyl, aryl, etc.), C (i.e., analkyl group, an aryl group, etc.), H, NR₂ (R is hydrogen, alkyl, aryl,etc.), or OR(R is alkyl or aryl). The phosphorus atom in an unmodifiedphosphate group is achiral. However, replacement of one of thenon-linking oxygens with one of the above atoms or groups of atomsrenders the phosphorus atom chiral; in other words a phosphorus atom ina phosphate group modified in this way is a stereogenic center. Thestereogenic phosphorus atom can possess either the “R” configuration(herein R_(P)) or the “S” configuration (herein S_(P)). Thus if 60% of apopulation of stereogenic phosphorus atoms have the R_(P) configuration,then the remaining 40% of the population of stereogenic phosphorus atomshave the S_(P) configuration.

In some embodiments, iRNA agents, having phosphate groups in which aphosphate non-linking oxygen has been replaced by another atom or groupof atoms, may contain a population of stereogenic phosphorus atoms inwhich at least about 50% of these atoms (e.g., at least about 60% ofthese atoms, at least about 70% of these atoms, at least about 80% ofthese atoms, at least about 90% of these atoms, at least about 95% ofthese atoms, at least about 98% of these atoms, at least about 99% ofthese atoms) have the S_(P) configuration. Alternatively, iRNA agentshaving phosphate groups in which a phosphate non-linking oxygen has beenreplaced by another atom or group of atoms may contain a population ofstereogenic phosphorus atoms in which at least about 50% of these atoms(e.g., at least about 60% of these atoms, at least about 70% of theseatoms, at least about 80% of these atoms, at least about 90% of theseatoms, at least about 95% of these atoms, at least about 98% of theseatoms, at least about 99% of these atoms) have the Rp configuration. Inother embodiments, the population of stereogenic phosphorus atoms mayhave the S_(P) configuration and may be substantially free ofstereogenic phosphorus atoms having the Rp configuration. In still otherembodiments, the population of stereogenic phosphorus atoms may have theR_(P) configuration and may be substantially free of stereogenicphosphorus atoms having the S_(P) configuration. As used herein, thephrase “substantially free of stereogenic phosphorus atoms having theR_(P) configuration” means that moieties containing stereogenicphosphorus atoms having the R_(P) configuration cannot be detected byconventional methods known in the art (chiral HPLC, ¹H NMR analysisusing chiral shift reagents, etc.). As used herein, the phrase“substantially free of stereogenic phosphorus atoms having the S_(P)configuration” means that moieties containing stereogenic phosphorusatoms having the S_(P) configuration cannot be detected by conventionalmethods known in the art (chiral HPLC, ¹H NMR analysis using chiralshift reagents, etc.).

In a preferred embodiment, modified iRNA agents contain aphosphorothioate group, i.e., a phosphate groups in which a phosphatenon-linking oxygen has been replaced by a sulfur atom. In an especiallypreferred embodiment, the population of phosphorothioate stereogenicphosphorus atoms may have the S_(P) configuration and be substantiallyfree of stereogenic phosphorus atoms having the R_(P) configuration.

Phosphorothioates may be incorporated into iRNA agents using dimerse.g., formulas X-1 and X-2. The former can be used to introducephosphorothioate

at the 3′ end of a strand, while the latter can be used to introducethis modification at the 5′ end or at a position that occurs e.g., 1, 2,3, 4, 5, or 6 nucleotides from either end of the strand. In the aboveformulas, Y can be 2-cyanoethoxy, W and Z can be O, R₂, can be, e.g., asubstituent that can impart the C-3 endo configuration to the sugar(e.g., OH, F, OCH₃), DMT is dimethoxytrityl, and “BASE” can be anatural, unusual, or a universal base.

X-1 and X-2 can be prepared using chiral reagents or directing groupsthat can result in phosphorothioate-containing dimers having apopulation of stereogenic phosphorus atoms having essentially only theR_(P) configuration (i.e., being substantially free of the S_(P)configuration) or only the S_(P) configuration (i.e., beingsubstantially free of the R_(P) configuration). Alternatively, dimerscan be prepared having a population of stereogenic phosphorus atoms inwhich about 50% of the atoms have the R_(P) configuration and about 50%of the atoms have the S_(P) configuration. Dimers having stereogenicphosphorus atoms with the R_(P) configuration can be identified andseparated from dimers having stereogenic phosphorus atoms with the S_(P)configuration using e.g., enzymatic degradation and/or conventionalchromatography techniques.

Cationic Groups

Modifications can also include attachment of one or more cationic groupsto the sugar, base, and/or the phosphorus atom of a phosphate ormodified phosphate backbone moiety. A cationic group can be attached toany atom capable of substitution on a natural, unusual or universalbase. A preferred position is one that does not interfere withhybridization, i.e., does not interfere with the hydrogen bondinginteractions needed for base pairing. A cationic group can be attachede.g., through the C2′ position of a sugar or analogous position in acyclic or acyclic sugar surrogate. Cationic groups can include e.g.,protonated amino groups, derived from e.g., O-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino);aminoalkoxy, e.g., O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, or amino acid); orNH(CH₂CH₂NH).CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino).

Nonphosphate Linkages

Modifications can also include the incorporation of nonphosphatelinkages at the 5′ and/or 3′ end of a strand. Examples of nonphosphatelinkages which can replace the phosphate group include methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl,carbamate, amide, thioether, ethylene oxide linker, sulfonate,sulfonamide, thioformacetal, formacetal, oxime, methyleneimino,methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo andmethyleneoxymethylimino. Preferred replacements include the methylphosphonate and hydroxylamino groups.

3′-bridging thiophosphates and 5′-bridging thiophosphates; locked-RNA,2′-5′ likages, inverted linkages, α-nucleosides; conjugate groups;abasic linkages; and 5′-phosphonates and 5′-phosphate prodrugs

Referring to formula X above, modifications can include replacement ofone of the bridging or linking phosphate oxygens in the phosphatebackbone moiety (W and Z). Unlike the situation where only one of X or Yis altered, the phosphorus center in the phosphorodithioates is achiralwhich precludes the formation of iRNA agents containing a stereogenicphosphorus atom.

Modifications can also include linking two sugars via a phosphate ormodified phosphate group through the 2′ position of a first sugar andthe 5′ position of a second sugar. Also contemplated are invertedlinkages in which both a first and second sugar are eached linkedthrough the respective 3′ positions. Modified RNA's can also include“abasic” sugars, which lack a nucleobase at C-1′. The sugar group canalso contain one or more carbons that possess the oppositestereochemical configuration than that of the corresponding carbon inribose. Thus, a modified iRNA agent can include nucleotides containinge.g., arabinose, as the sugar. In another subset of this modification,the natural, unusual, or universal base may have the a-configuration.Modifcations can also include L-RNA.

Modifications can also include 5′-phosphonates, e.g.,P(O)(O⁻)₂—X—C⁵′-sugar (X═CH₂, CF₂, CHF and 5′-phosphate prodrugs, e.g.,P(O)[OCH₂CH₂SC(O)R]₂CH₂C⁵-sugar. In the latter case, the prodrug groupsmay be decomposed via reaction first with carboxy esterases. Theremaining ethyl thiolate group via intramolecular S_(N2) displacementcan depart as episulfide to afford the underivatized phosphate group.

Modification can also include the addition of conjugating groupsdescribed elseqhere herein, which are prefereably attached to an iRNAagent through any amino group available for conjugation.

Nuclease resistant modifications include some which can be placed onlyat the terminus and others which can go at any position. Generally themodifications that can inhibit hybridization so it is preferably to usethem only in terminal regions, and preferrable to not use them at thecleavage site or in the cleavage region of an sequence which targets asubject sequence or gene. The can be used anywhere in a sense sequence,provided that sufficient hybridization between the two sequences of theiRNA agent is maintained. In some embodiments it is desirable to put theNRM at the cleavage site or in the cleavage region of a sequence whichdoes not target a subject sequence or gene, as it can minimizeoff-target silencing.

In addition, an iRNA agent described herein can have an overhang whichdoes not form a duplex structure with the other sequence of the iRNAagent-it is an overhang, but it does hybridize, either with itself, orwith another nucleic acid, other than the other sequence of the iRNAagent.

In most cases, the nuclease-resistance promoting modifications will bedistributed differently depending on whether the sequence will target asequence in the subject (often referred to as an anti-sense sequence) orwill not target a sequence in the subject (often referred to as a sensesequence). If a sequence is to target a sequence in the subject,modifications which interfer with or inhibit endonuclease cleavageshould not be inserted in the region which is subject to RISC mediatedcleavage, e.g., the cleavage site or the cleavage region (As describedin Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated byreference, cleavage of the target occurs about in the middle of a 20 or21 nt guide RNA, or about 10 or 11 nucleotides upstream of the firstnucleotide which is complementary to the guide sequence. As used hereincleavage site refers to the nucleotide on either side of the cleavagesite, on the target or on the iRNA agent strand which hybridizes to it.Cleavage region means an nucleotide with 1, 2, or 3 nucletides of thecleave site, in either direction.)

Such modifications can be introduced into the terminal regions, e.g., atthe terminal position or with 2, 3, 4, or 5 positions of the terminus,of a sequence which targets or a sequence which does not target asequence in the subject.

An iRNA agent can have a first and a second strand chosen from thefollowing:

a first strand which does not target a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which does not target a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a first strand which does not target a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a first strand which does not target a sequence and which has an NRMmodification at the cleavage site or in the cleavage region;

a first strand which does not target a sequence and which has an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end; and

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end(5′ end NRM modifications are preferentially not at the terminus butrather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of anantisense strand);

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a second strand which targets a sequence and which preferably does nothave an an NRM modification at the cleavage site or in the cleavageregion;

a second strand which targets a sequence and which does not have an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end(5′ end NRMmodifications are preferentially not at the terminus but rather at aposition 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisensestrand).

An iRNA agent can also target two sequences and can have a first andsecond strand chosen from:

a first strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end(5′ end NRM modifications are preferentially not at the terminus butrather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of anantisense strand);

a first strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a first strand which targets a sequence and which preferably does nothave an an NRM modification at the cleavage site or in the cleavageregion;

a first strand which targets a sequence and which dose not have an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end (5′ end NRMmodifications are preferentially not at the terminus but rather at aposition 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisensestrand) and

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end(5′ end NRM modifications are preferentially not at the terminus butrather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of anantisense strand);

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a second strand which targets a sequence and which preferably does nothave an an NRM modification at the cleavage site or in the cleavageregion; a second strand which targets a sequence and which dose not havean NRM modification at the cleavage site or in the cleavage region andone or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4,5, or 6 positions from the 5′ end, or NRM modifications at or within 1,2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end (5′ end NRMmodifications are preferentially not at the terminus but rather at aposition 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisensestrand).

Ribose Mimics

The monomers and methods described herein can be used to prepare an RNA,e.g., an iRNA agent, that incorporates a ribose mimic, such as thosedescribed herein and those described in copending co-owned U.S.Provisional Application Ser. No. 60/454,962, filed on Mar. 13, 2003, andInternational Application No. PCT/US04/07070, both of which are herebyincorporated by reference.

Thus, an aspect of the invention features an iRNA agent that includes asecondary hydroxyl group, which can increase efficacy and/or confernuclease resistance to the agent. Nucleases, e.g., cellular nucleases,can hydrolyze nucleic acid phosphodiester bonds, resulting in partial orcomplete degradation of the nucleic acid. The secondary hydroxy groupconfers nuclease resistance to an iRNA agent by rendering the iRNA agentless prone to nuclease degradation relative to an iRNA which lacks themodification. While not wishing to be bound by theory, it is believedthat the presence of a secondary hydroxyl group on the iRNA agent canact as a structural mimic of a 3′ ribose hydroxyl group, thereby causingit to be less susceptible to degradation.

The secondary hydroxyl group refers to an “OH” radical that is attachedto a carbon atom substituted by two other carbons and a hydrogen. Thesecondary hydroxyl group that confers nuclease resistance as describedabove can be part of any acyclic carbon-containing group. The hydroxylmay also be part of any cyclic carbon-containing group, and preferablyone or more of the following conditions is met (1) there is no ribosemoiety between the hydroxyl group and the terminal phosphate group or(2) the hydroxyl group is not on a sugar moiety which is coupled to abase. The hydroxyl group is located at least two bonds (e.g., at leastthree bonds away, at least four bonds away, at least five bonds away, atleast six bonds away, at least seven bonds away, at least eight bondsaway, at least nine bonds away, at least ten bonds away, etc.) from theterminal phosphate group phosphorus of the iRNA agent. In preferredembodiments, there are five intervening bonds between the terminalphosphate group phosphorus and the secondary hydroxyl group.

Preferred iRNA agent delivery modules with five intervening bondsbetween the terminal phosphate group phosphorus and the secondaryhydroxyl group have the following structure (see formula Y below):

Referring to formula Y, A is an iRNA agent, including any iRNA agentdescribed herein. The iRNA agent may be connected directly or indirectly(e.g., through a spacer or linker) to “W” of the phosphate group. Thesespacers or linkers can include e.g., —(CH₂)_(n)—, —(CH₂)_(n)N—,—(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g., n=3 or 6),abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether,disulfide, thiourea, sulfonamide, or morpholino, or biotin andfluorescein reagents.

The iRNA agents can have a terminal phosphate group that is unmodified(e.g., W, X, Y, and Z are O) or modified. In a modified phosphate group,W and Z can be independently NH, O, or S; and X and Y can beindependently S, Se, BH₃ ⁻, C₁-C₆ alkyl, C₆-C₁₀ aryl, H, O, O⁻, alkoxyor amino (including alkylamino, arylamino, etc.). Preferably, W, X and Zare O and Y is S.

R₁ and R₃ are each, independently, hydrogen; or C₁-C₁₀₀ alkyl,optionally substituted with hydroxyl, amino, halo, phosphate or sulfateand/or may be optionally inserted with N, O, S, alkenyl or alkynyl.

R₂ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl; or, when n is 1, R₂ may be taken togetherwith R₄ or R₆ to form a ring of 5-12 atoms.

R₄ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl; or, when n is 1, R₄ may be taken togetherwith R₂ or R₅ to form a ring of 5-12 atoms.

R₅ is hydrogen, C₁-C₁₀₀ alkyl optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl; or, when n is 1, R₅ may be taken togetherwith R₄ to form a ring of 5-12 atoms.

R₆ is hydrogen, C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl, or, when n is 1, R₆ may be taken togetherwith R₂ to form a ring of 6-10 atoms;

R₇ is hydrogen, C₁-C₁₀₀ alkyl, or C(O)(CH₂)_(q)C(O)NHR₉; T is hydrogenor a functional group; n and q are each independently 1-100; R₈ isC₁-C₁₀ alkyl or C₆-C₁₀ aryl; and R₉ is hydrogen, C1-C10 alkyl, C₆-C₁₀aryl or a solid support agent.

Preferred embodiments may include one of more of the following subsetsof iRNA agent delivery modules.

In one subset of RNAi agent delivery modules, A can be connecteddirectly or indirectly through a terminal 3′ or 5′ ribose sugar carbonof the RNA agent.

In another subset of RNAi agent delivery modules, X, W, and Z are O andY is S.

In still yet another subset of RNAi agent delivery modules, n is 1, andR₂ and R₆ are taken together to form a ring containing six atoms and R₄and R₅ are taken together to form a ring containing six atoms.Preferably, the ring system is a trans-decalin. For example, the RNAiagent delivery module of this subset can include a compound of Formula(Y-1):

The functional group can be, for example, a targeting group (e.g., asteroid or a carbohydrate), a reporter group (e.g., a fluorophore), or alabel (an isotopically labelled moiety). The targeting group can furtherinclude protein binding agents, endothelial cell targeting groups (e.g.,RGD peptides and mimetics), cancer cell targeting groups (e.g., folateVitamin B12, Biotin), bone cell targeting groups (e.g., bisphosphonates,polyglutamates, polyaspartates), multivalent mannose (for e.g.,macrophage testing), lactose, galactose, N-acetyl-galactosamine,monoclonal antibodies, glycoproteins, lectins, melanotropin, orthyrotropin.

As can be appreciated by the skilled artisan, methods of synthesizingthe compounds of the formulae herein will be evident to those ofordinary skill in the art. The synthesized compounds can be separatedfrom a reaction mixture and further purified by a method such as columnchromatography, high pressure liquid chromatography, orrecrystallization. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

Palindromes

The monomers and methods described herein can be used to prepare an RNA,e.g., an iRNA agent, having a palindrome structure as described hereinand those described in one or more of U.S. Provisional Application Ser.No. 60/452,682, filed Mar. 7, 2003; U.S. Provisional Application Ser.No. 60/462,894, filed Apr. 14, 2003; and International Application No.PCT/US04/07070, filed Mar. 8, 2004, all of which are hereby incorporatedby reference. The iRNA agents of the invention can target more than oneRNA region. For example, an iRNA agent can include a first and secondsequence that are sufficiently complementary to each other to hybridize.The first sequence can be complementary to a first target RNA region andthe second sequence can be complementary to a second target RNA region.The first and second sequences of the iRNA agent can be on different RNAstrands, and the mismatch between the first and second sequences can beless than 50%, 40%, 30%, 20%, 10%, 5%, or 1%. The first and secondsequences of the iRNA agent are on the same RNA strand, and in a relatedembodiment more than 50%, 60%, 70%, 80%, 90%, 95%, or 1% of the iRNAagent can be in bimolecular form. The first and second sequences of theiRNA agent can be fully complementary to each other.

The first target RNA region can be encoded by a first gene and thesecond target RNA region can encoded by a second gene, or the first andsecond target RNA regions can be different regions of an RNA from asingle gene. The first and second sequences can differ by at least 1nucleotide.

The first and second target RNA regions can be on transcripts encoded byfirst and second sequence variants, e.g., first and second alleles, of agene. The sequence variants can be mutations, or polymorphisms, forexample. The first target RNA region can include a nucleotidesubstitution, insertion, or deletion relative to the second target RNAregion, or the second target RNA region can a mutant or variant of thefirst target region.

The first and second target RNA regions can comprise viral or human RNAregions. The first and second target RNA regions can also be on varianttranscripts of an oncogene or include different mutations of a tumorsuppressor gene transcript. In addition, the first and second target RNAregions can correspond to hot-spots for genetic variation.

The compositions of the invention can include mixtures of iRNA agentmolecules. For example, one iRNA agent can contain a first sequence anda second sequence sufficiently complementary to each other to hybridize,and in addition the first sequence is complementary to a first targetRNA region and the second sequence is complementary to a second targetRNA region. The mixture can also include at least one additional iRNAagent variety that includes a third sequence and a fourth sequencesufficiently complementary to each other to hybridize, and where thethird sequence is complementary to a third target RNA region and thefourth sequence is complementary to a fourth target RNA region. Inaddition, the first or second sequence can be sufficiently complementaryto the third or fourth sequence to be capable of hybridizing to eachother. The first and second sequences can be on the same or differentRNA strands, and the third and fourth sequences can be on the same ordifferent RNA strands.

The target RNA regions can be variant sequences of a viral or human RNA,and in certain embodiments, at least two of the target RNA regions canbe on variant transcripts of an oncogene or tumor suppressor gene. Thetarget RNA regions can correspond to genetic hot-spots.

Methods of making an iRNA agent composition can include obtaining orproviding information about a region of an RNA of a target gene (e.g., aviral or human gene, or an oncogene or tumor suppressor, e.g., p53),where the region has high variability or mutational frequency (e.g., inhumans). In addition, information about a plurality of RNA targetswithin the region can be obtained or provided, where each RNA targetcorresponds to a different variant or mutant of the gene (e.g., a regionincluding the codon encoding p53 248Q and/or p53 249S).

The iRNA agent can be constructed such that a first sequence iscomplementary to a first of the plurality of variant RNA targets (e.g.,encoding 249Q) and a second sequence is complementary to a second of theplurality of variant RNA targets (e.g., encoding 249S), and the firstand second sequences can be sufficiently complementary to hybridize.Sequence analysis, e.g., to identify common mutants in the target gene,can be used to identify a region of the target gene that has highvariability or mutational frequency. A region of the target gene havinghigh variability or mutational frequency can be identified by obtainingor providing genotype information about the target gene from apopulation.

Expression of a target gene can be modulated, e.g., downregulated orsilenced, by providing an iRNA agent that has a first sequence and asecond sequence sufficiently complementary to each other to hybridize.In addition, the first sequence can be complementary to a first targetRNA region and the second sequence can be complementary to a secondtarget RNA region.

An iRNA agent can include a first sequence complementary to a firstvariant RNA target region and a second sequence complementary to asecond variant RNA target region. The first and second variant RNAtarget regions can correspond to first and second variants or mutants ofa target gene, e.g., viral gene, tumor suppressor or oncogene. The firstand second variant target RNA regions can include allelic variants,mutations (e.g., point mutations), or polymorphisms of the target gene.The first and second variant RNA target regions can correspond togenetic hot-spots.

A plurality of iRNA agents (e.g., a panel or bank) can be provided.

Other than Canonical Watson-Crick Duplex Structures

The monomers and methods described herein can be used to prepare an RNA,e.g., an iRNA agent, having monomers which can form other than acanonical Watson-Crick pairing with another monomer, e.g., a monomer onanother strand, such as those described herein and those described inU.S. Provisional Application Ser. No. 60/465,665, filed Apr. 25, 2003,and International Application No. PCT/US04/07070, filed Mar. 8, 2004,both of which are hereby incorporated by reference.

The use of “other than canonical Watson-Crick pairing” between monomersof a duplex can be used to control, often to promote, melting of all orpart of a duplex. The iRNA agent can include a monomer at a selected orconstrained position that results in a first level of stability in theiRNA agent duplex (e.g., between the two separate molecules of a doublestranded iRNA agent) and a second level of stability in a duplex betweena sequence of an iRNA agent and another sequence molecule, e.g., atarget or off-target sequence in a subject. In some cases the secondduplex has a relatively greater level of stability, e.g., in a duplexbetween an anti-sense sequence of an iRNA agent and a target mRNA. Inthis case one or more of the monomers, the position of the monomers inthe iRNA agent, and the target sequence (sometimes referred to herein asthe selection or constraint parameters), are selected such that the iRNAagent duplex is has a comparatively lower free energy of association(which while not wishing to be bound by mechanism or theory, is believedto contribute to efficacy by promoting disassociation of the duplex iRNAagent in the context of the RISC) while the duplex formed between ananti-sense targeting sequence and its target sequence, has a relativelyhigher free energy of association (which while not wishing to be boundby mechanism or theory, is believed to contribute to efficacy bypromoting association of the anti-sense sequence and the target RNA).

In other cases the second duplex has a relatively lower level ofstability, e.g., in a duplex between a sense sequence of an iRNA agentand an off-target mRNA. In this case one or more of the monomers, theposition of the monomers in the iRNA agent, and an off-target sequence,are selected such that the iRNA agent duplex is has a comparativelyhigher free energy of association while the duplex formed between asense targeting sequence and its off-target sequence, has a relativelylower free energy of association (which while not wishing to be bound bymechanism or theory, is believed to reduce the level of off-targetsilencing by contribute to efficacy by promoting disassociation of theduplex formed by the sense strand and the off-target sequence).

Thus, inherent in the structure of the iRNA agent is the property ofhaving a first stability for the intra-iRNA agent duplex and a secondstability for a duplex formed between a sequence from the iRNA agent andanother RNA, e.g., a target mRNA. As discussed above, this can beaccomplished by judicious selection of one or more of the monomers at aselected or constrained position, the selection of the position in theduplex to place the selected or constrained position, and selection ofthe sequence of a target sequence (e.g., the particular region of atarget gene which is to be targeted). The iRNA agent sequences whichsatisfy these requirements are sometimes referred herein as constrainedsequences. Exercise of the constraint or selection parameters can e,e.g., by inspection, or by computer assisted methods. Exercise of theparameters can result in selection of a target sequence and ofparticular monomers to give a desired result in terms of the stability,or relative stability, of a duplex.

Thus, in another aspect, the invention features, an iRNA agent whichincludes: a first sequence which targets a first target region and asecond sequence which targets a second target region. The first andsecond sequences have sufficient complementarity to each other tohybridize, e.g., under physiological conditions, e.g., underphysiological conditions but not in contact with a helicase or otherunwinding enzyme. In a duplex region of the iRNA agent, at a selected orconstrained position, the first target region has a first monomer, andthe second target region has a second monomer. The first and secondmonomers occupy complementary or corresponding positions. One, andpreferably both monomers are selected such that the stability of thepairing of the monomers contribute to a duplex between the first andsecond sequence will differ form the stability of the pairing betweenthe first or second sequence with a target sequence.

Usually, the monomers will be selected (selection of the target sequencemay be required as well) such that they form a pairing in the iRNA agentduplex which has a lower free energy of dissociation, and a lower Tm,than will be possessed by the paring of the monomer with itscomplementary monomer in a duplex between the iRNA agent sequence and atarget RNA duplex.

The constraint placed upon the monomers can be applied at a selectedsite or at more than one selected site. By way of example, theconstraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7sites in an iRNA agent duplex.

A constrained or selected site can be present at a number of positionsin the iRNA agent duplex. E.g., a constrained or selected site can bepresent within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of aduplexed sequence. A constrained or selected site can be present in themiddle of the duplex region, e.g., it can be more than 3, 4, 5, or 6,positions from the end of a duplexed region.

In some embodiment the duplex region of the iRNA agent will have,mismatches, in addition to the selected or constrained site or sites.Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which donot form canonical Watson-Crick pairs or which do not hybridize.Overhangs are discussed in detail elsewhere herein but are preferablyabout 2 nucleotides in length. The overhangs can be complementary to thegene sequences being targeted or can be other sequence. TT is apreferred overhang sequence. The first and second iRNA agent sequencescan also be joined, e.g., by additional bases to form a hairpin, or byother non-base linkers.

The monomers can be selected such that: first and second monomers arenaturally occurring ribonuceotides, or modified ribonucleotides havingnaturally occurring bases, and when occupying complemetary sites eitherdo not pair and have no substantial level of H-bonding, or form a noncanonical Watson-Crick pairing and form a non-canonical pattern of Hbonding, which usually have a lower free energy of dissociation thanseen in a canonical Watson-Crick pairing, or otherwise pair to give afree energy of association which is less than that of a preselectedvalue or is less, e.g., than that of a canonical pairing. When one (orboth) of the iRNA agent sequences duplexes with a target, the first (orsecond) monomer forms a canonical Watson-Crick pairing with the base inthe complemetary position on the target, or forms a non canonicalWatson-Crick pairing having a higher free energy of dissociation and ahigher Tm than seen in the paring in the iRNA agent. The classicalWatson-Crick parings are as follows: A-T, G-C, and A-U. Non-canonicalWatson-Crick pairings are known in the art and can include, U—U, G-G,G-A_(trans), &A_(cis), and GU.

The monomer in one or both of the sequences is selected such that, itdoes not pair, or forms a pair with its corresponding monomer in theother sequence which minimizes stability (e.g., the H bonding formedbetween the monomer at the selected site in the one sequence and itsmonomer at the corresponding site in the other sequence are less stablethan the H bonds formed by the monomer one (or both) of the sequenceswith the respective target sequence. The monomer is one or both strandsis also chosen to promote stability in one or both of the duplexes madeby a strand and its target sequence. E.g., one or more of the monomersand the target sequences are selected such that at the selected orconstrained position, there is are no H bonds formed, or a non canonicalpairing is formed in the iRNA agent duplex, or otherwise they otherwisepair to give a free energy of association which is less than that of apreselected value or is less, e.g., than that of a canonical pairing,but when one (or both) sequences form a duplex with the respectivetarget, the pairing at the selected or constrained site is a canonicalWatson-Crick paring.

The inclusion of such a monomers will have one or more of the followingeffects: it will destabilize the iRNA agent duplex, it will destabilizeinteractions between the sense sequence and unintended target sequences,sometimes referred to as off-target sequences, and duplex interactionsbetween the a sequence and the intended target will not be destabilized.

By way of example:

The monomer at the selected site in the first sequence includes an A (ora modified base which pairs with T), and the monomer in at the selectedposition in the second sequence is chosen from a monomer which will notpair or which will form a non-canonical pairing, e.g., G. These will beuseful in applications wherein the target sequence for the firstsequence has a T at the selected position. In embodiments where bothtarget duplexes are stabilized it is useful wherein the target sequencefor the second strand has a monomer which will form a canonicalWatson-Crick pairing with the monomer selected for the selected positionin the second strand.

The monomer at the selected site in the first sequence includes U (or amodified base which pairs with A), and the monomer in at the selectedposition in the second sequence is chosen from a monomer which will notpair or which will form a non-canonical pairing, e.g., U or G. Thesewill be useful in applications wherein the target sequence for the firstsequence has a T at the selected position. In embodiments where bothtarget duplexes are stabilized it is useful wherein the target sequencefor the second strand has a monomer which will form a canonicalWatson-Crick pairing with the monomer selected for the selected positionin the second strand.

The monomer at the selected site in the first sequence includes a G (ora modified base which pairs with C), and the monomer in at the selectedposition in the second sequence is chosen from a monomer which will notpair or which will form a non-canonical pairing, e.g., G, A_(cis),A_(trans), or U. These will be useful in applications wherein the targetsequence for the first sequence has a T at the selected position. Inembodiments where both target duplexes are stabilized it is usefulwherein the target sequence for the second strand has a monomer whichwill form a canonical Watson-Crick pairing with the monomer selected forthe selected position in the second strand.

The monomer at the selected site in the first sequence includes a C (ora modified base which pairs with G), and the monomer in at the selectedposition in the second sequence is chosen a monomer which will not pairor which will form a non-canonical pairing. These will be useful inapplications wherein the target sequence for the first sequence has a Tat the selected position. In embodiments where both target duplexes arestabilized it is useful wherein the target sequence for the secondstrand has a monomer which will form a canonical Watson-Crick pairingwith the monomer selected for the selected position in the secondstrand.

A non-naturally occurring or modified monomer or monomers can be chosensuch that when a non-naturally occurring or modified monomer occupies apositions at the selected or constrained position in an iRNA agent theyexhibit a first free energy of dissociation and when one (or both) ofthem pairs with a naturally occurring monomer, the pair exhibits asecond free energy of dissociation, which is usually higher than that ofthe pairing of the first and second monomers. E.g., when the first andsecond monomers occupy complementary positions they either do not pairand have no substantial level of H-bonding, or form a weaker bond thanone of them would form with a naturally occurring monomer, and reducethe stability of that duplex, but when the duplex dissociates at leastone of the strands will form a duplex with a target in which theselected monomer will promote stability, e.g., the monomer will form amore stable pair with a naturally occurring monomer in the targetsequence than the pairing it formed in the iRNA agent.

An example of such a pairing is 2-amino A and either of a 2-thiopyrimidine analog of U or T.

When placed in complementary positions of the iRNA agent these monomerswill pair very poorly and will minimize stability. However, a duplex isformed between 2 amino A and the U of a naturally occurring target, or aduplex is between 2-thio U and the A of a naturally occurring target or2-thio T and the A of a naturally occurring target will have arelatively higher free energy of dissociation and be more stable. Thisis shown in the FIG. 12.

The pair shown in FIG. 12 (the 2-amino A and the 2-s U and T) isexemplary. In another embodiment, the monomer at the selected positionin the sense strand can be a universal pairing moiety. A universalpairing agent will form some level of H bonding with more than one andpreferably all other naturally occurring monomers. An examples of auniversal pairing moiety is a monomer which includes 3-nitro pyrrole.(Examples of other candidate universal base analogs can be found in theart, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated byreference. Examples can also be found in the section on Universal Basesbelow.) In these cases the monomer at the corresponding position of theanti-sense strand can be chosen for its ability to form a duplex withthe target and can include, e.g., A, U, G, or C.

iRNA agents of the invention can include:

A sense sequence, which preferably does not target a sequence in asubject, and an anti-sense sequence, which targets a target gene in asubject. The sense and anti-sense sequences have sufficientcomplementarity to each other to hybridize hybridize, e.g., underphysiological conditions, e.g., under physiological conditions but notin contact with a helicase or other unwinding enzyme. In a duplex regionof the iRNA agent, at a selected or constrained position, the monomersare selected such that:

The monomer in the sense sequence is selected such that, it does notpair, or forms a pair with its corresponding monomer in the anti-sensestrand which minimizes stability (e.g., the H bonding formed between themonomer at the selected site in the sense strand and its monomer at thecorresponding site in the anti-sense strand are less stable than the Hbonds formed by the monomer of the anti-sense sequence and its canonicalWatson-Crick partner or, if the monomer in the anti-sense strandincludes a modified base, the natural analog of the modified base andits canonical Watson-Crick partner).

The monomer is in the corresponding position in the anti-sense strand isselected such that it maximizes the stability of a duplex it forms withthe target sequence, e.g., it forms a canonical Watson-Crick paring withthe monomer in the corresponding position on the target stand;

Optionally, the monomer in the sense sequence is selected such that, itdoes not pair, or forms a pair with its corresponding monomer in theanti-sense strand which minimizes stability with an off-target sequence.

The inclusion of such a monomers will have one or more of the followingeffects: it will destabilize the iRNA agent duplex, it will destabilizeinteractions between the sense sequence and unintended target sequences,sometimes referred to as off-target sequences, and duplex interactionsbetween the anti-sense strand and the intended target will not bedestabilized.

The constraint placed upon the monomers can be applied at a selectedsite or at more than one selected site. By way of example, theconstraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7sites in an iRNA agent duplex.

A constrained or selected site can be present at a number of positionsin the iRNA agent duplex. E.g., a constrained or selected site can bepresent within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of aduplexed sequence. A constrained or selected site can be present in themiddle of the duplex region, e.g., it can be more than 3, 4, 5, or 6,positions from the end of a duplexed region.

In some embodiment the duplex region of the iRNA agent will have,mismatches, in addition to the selected or constrained site or sites.Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which donot form canonical Watson-Crick pairs or which do not hybridize.Overhangs are discussed in detail elsewhere herein but are preferablyabout 2 nucleotides in length. The overhangs can be complementary to thegene sequences being targeted or can be other sequence. TT is apreferred overhang sequence. The first and second iRNA agent sequencescan also be joined, e.g., by additional bases to form a hairpin, or byother non-base linkers.

The monomers can be selected such that: first and second monomers arenaturally occurring ribonuceotides, or modified ribonucleotides havingnaturally occurring bases, and when occupying complemetary sites eitherdo not pair and have no substantial level of H-bonding, or form a noncanonical Watson-Crick pairing and form a non-canonical pattern of Hbonding, which usually have a lower free energy of dissociation thanseen in a canonical Watson-Crick pairing, or otherwise pair to give afree energy of association which is less than that of a preselectedvalue or is less, e.g., than that of a canonical pairing. When one (orboth) of the iRNA agent sequences duplexes with a target, the first (orsecond) monomer forms a canonical Watson-Crick pairing with the base inthe complemetary position on the target, or forms a non canonicalWatson-Crick pairing having a higher free energy of dissociation and ahigher Tm than seen in the paring in the iRNA agent. The classicalWatson-Crick parings are as follows: A-T, G-C, and A-U. Non-canonicalWatson-Crick pairings are known in the art and can include, U—U, G-G,G-A_(trans), &A_(cts), and GU.

The monomer in one or both of the sequences is selected such that, itdoes not pair, or forms a pair with its corresponding monomer in theother sequence which minimizes stability (e.g., the H bonding formedbetween the monomer at the selected site in the one sequence and itsmonomer at the corresponding site in the other sequence are less stablethan the H bonds formed by the monomer one (or both) of the sequenceswith the respective target sequence. The monomer is one or both strandsis also chosen to promote stability in one or both of the duplexes madeby a strand and its target sequence. E.g., one or more of the monomersand the target sequences are selected such that at the selected orconstrained position, there is are no H bonds formed, or a non canonicalpairing is formed in the iRNA agent duplex, or otherwise they otherwisepair to give a free energy of association which is less than that of apreselected value or is less, e.g., than that of a canonical pairing,but when one (or both) sequences form a duplex with the respectivetarget, the pairing at the selected or constrained site is a canonicalWatson-Crick paring.

The inclusion of such a monomers will have one or more of the followingeffects: it will destabilize the iRNA agent duplex, it will destabilizeinteractions between the sense sequence and unintended target sequences,sometimes referred to as off-target sequences, and duplex interactionsbetween the a sequence and the intended target will not be destabilized.

By way of example:

The monomer at the selected site in the first sequence includes an A (ora modified base which pairs with T), and the monomer in at the selectedposition in the second sequence is chosen from a monomer which will notpair or which will form a non-canonical pairing, e.g., G. These will beuseful in applications wherein the target sequence for the firstsequence has a T at the selected position. In embodiments where bothtarget duplexes are stabilized it is useful wherein the target sequencefor the second strand has a monomer which will form a canonicalWatson-Crick pairing with the monomer selected for the selected positionin the second strand.

The monomer at the selected site in the first sequence includes U (or amodified base which pairs with A), and the monomer in at the selectedposition in the second sequence is chosen from a monomer which will notpair or which will form a non-canonical pairing, e.g., U or G. Thesewill be useful in applications wherein the target sequence for the firstsequence has a T at the selected position. In embodiments where bothtarget duplexes are stabilized it is useful wherein the target sequencefor the second strand has a monomer which will form a canonicalWatson-Crick pairing with the monomer selected for the selected positionin the second strand.

The monomer at the selected site in the first sequence includes a G (ora modified base which pairs with C), and the monomer in at the selectedposition in the second sequence is chosen from a monomer which will notpair or which will form a non-canonical pairing, e.g., G, A_(cis),A_(trans), or U. These will be useful in applications wherein the targetsequence for the first sequence has a T at the selected position. Inembodiments where both target duplexes are stabilized it is usefulwherein the target sequence for the second strand has a monomer whichwill form a canonical Watson-Crick pairing with the monomer selected forthe selected position in the second strand.

The monomer at the selected site in the first sequence includes a C (ora modified base which pairs with G), and the monomer in at the selectedposition in the second sequence is chosen a monomer which will not pairor which will form a non-canonical pairing. These will be useful inapplications wherein the target sequence for the first sequence has a Tat the selected position. In embodiments where both target duplexes arestabilized it is useful wherein the target sequence for the secondstrand has a monomer which will form a canonical Watson-Crick pairingwith the monomer selected for the selected position in the secondstrand.

A non-naturally occurring or modified monomer or monomers can be chosensuch that when a non-naturally occurring or modified monomer occupies apositions at the selected or constrained position in an iRNA agent theyexhibit a first free energy of dissociation and when one (or both) ofthem pairs with a naturally occurring monomer, the pair exhibits asecond free energy of dissociation, which is usually higher than that ofthe pairing of the first and second monomers. E.g., when the first andsecond monomers occupy complementary positions they either do not pairand have no substantial level of H-bonding, or form a weaker bond thanone of them would form with a naturally occurring monomer, and reducethe stability of that duplex, but when the duplex dissociates at leastone of the strands will form a duplex with a target in which theselected monomer will promote stability, e.g., the monomer will form amore stable pair with a naturally occurring monomer in the targetsequence than the pairing it formed in the iRNA agent.

An example of such a pairing is 2-amino A and either of a 2-thiopyrimidine analog of U or T.

When placed in complementary positions of the iRNA agent these monomerswill pair very poorly and will minimize stability. However, a duplex isformed between 2 amino A and the U of a naturally occurring target, or aduplex is between 2-thio U and the A of a naturally occurring target or2-thio T and the A of a naturally occurring target will have arelatively higher free energy of dissociation and be more stable.

The monomer at the selected position in the sense strand can be auniversal pairing moiety. A universal pairing agent will form some levelof H bonding with more than one and preferably all other naturallyoccurring monomers. An examples of a universal pairing moiety is amonomer which includes 3-nitro pyrrole. (Examples of other candidateuniversal base analogs can be found in the art, e.g., in Loakes, 2001,NAR 29: 2437-2447, hereby incorporated by reference. Examples can alsobe found in the section on Universal Bases below.) In these cases themonomer at the corresponding position of the anti-sense strand can bechosen for its ability to form a duplex with the target and can include,e.g., A, U, G, or C.

iRNA agents of the invention can include:

A sense sequence, which preferably does not target a sequence in asubject, and an anti-sense sequence, which targets a target gene in asubject. The sense and anti-sense sequences have sufficientcomplementarity to each other to hybridize hybridize, e.g., underphysiological conditions, e.g., under physiological conditions but notin contact with a helicase or other unwinding enzyme. In a duplex regionof the iRNA agent, at a selected or constrained position, the monomersare selected such that:

The monomer in the sense sequence is selected such that, it does notpair, or forms a pair with its corresponding monomer in the anti-sensestrand which minimizes stability (e.g., the H bonding formed between themonomer at the selected site in the sense strand and its monomer at thecorresponding site in the anti-sense strand are less stable than the Hbonds formed by the monomer of the anti-sense sequence and its canonicalWatson-Crick partner or, if the monomer in the anti-sense strandincludes a modified base, the natural analog of the modified base andits canonical Watson-Crick partner);

The monomer is in the corresponding position in the anti-sense strand isselected such that it maximizes the stability of a duplex it forms withthe target sequence, e.g., it forms a canonical Watson-Crick paring withthe monomer in the corresponding position on the target stand;

Optionally, the monomer in the sense sequence is selected such that, itdoes not pair, or forms a pair with its corresponding monomer in theanti-sense strand which minimizes stability with an off-target sequence.

The inclusion of such a monomers will have one or more of the followingeffects: it will destabilize the iRNA agent duplex, it will destabilizeinteractions between the sense sequence and unintended target sequences,sometimes referred to as off-target sequences, and duplex interactionsbetween the anti-sense strand and the intended target will not bedestabilized.

The constraint placed upon the monomers can be applied at a selectedsite or at more than one selected site. By way of example, theconstraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7sites in an iRNA agent duplex.

A constrained or selected site can be present at a number of positionsin the iRNA agent duplex. E.g., a constrained or selected site can bepresent within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of aduplexed sequence. A constrained or selected site can be present in themiddle of the duplex region, e.g., it can be more than 3, 4, 5, or 6,positions from the end of a duplexed region.

The iRNA agent can be selected to target a broad spectrum of genes,including any of the genes described herein.

In a preferred embodiment the iRNA agent has an architecture(architecture refers to one or more of overall length, length of aduplex region, the presence, number, location, or length of overhangs,sing strand versus double strand form) described herein.

E.g., the iRNA agent can be less than 30 nucleotides in length, e.g.,21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length andthere is a duplex region of about 19 pairs. In one embodiment, the iRNAis 21 nucleotides in length, and the duplex region of the iRNA is 19nucleotides. In another embodiment, the iRNA is greater than 30nucleotides in length.

In some embodiment the duplex region of the iRNA agent will have,mismatches, in addition to the selected or constrained site or sites.Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which donot form canonical Watson-Crick pairs or which do not hybridize.Overhangs are discussed in detail elsewhere herein but are preferablyabout 2 nucleotides in length. The overhangs can be complementary to thegene sequences being targeted or can be other sequence. TT is apreferred overhang sequence. The first and second iRNA agent sequencescan also be joined, e.g., by additional bases to form a hairpin, or byother non-base linkers.

One or more selection or constraint parameters can be exercised suchthat: monomers at the selected site in the sense and anti-sensesequences are both naturally occurring ribonucleotides, or modifiedribonucleotides having naturally occurring bases, and when occupyingcomplementary sites in the iRNA agent duplex either do not pair and haveno substantial level of H-bonding, or form a non-canonical Watson-Crickpairing and thus form a non-canonical pattern of H bonding, whichgenerally have a lower free energy of dissociation than seen in aWatson-Crick pairing, or otherwise pair to give a free energy ofassociation which is less than that of a preselected value or is less,e.g., than that of a canonical pairing. When one, usually the anti-sensesequence of the iRNA agent sequences forms a duplex with anothersequence, generally a sequence in the subject, and generally a targetsequence, the monomer forms a classic Watson-Crick pairing with the basein the complementary position on the target, or forms a non-canonicalWatson-Crick pairing having a higher free energy of dissociation and ahigher Tm than seen in the paring in the iRNA agent. Optionally, whenthe other sequence of the iRNA agent, usually the sense sequences formsa duplex with another sequence, generally a sequence in the subject, andgenerally an off-target sequence, the monomer fails to forms a canonicalWatson-Crick pairing with the base in the complementary position on theoff target sequence, e.g., it forms or forms a non-canonicalWatson-Crick pairing having a lower free energy of dissociation and alower Tm.

By way of example:

the monomer at the selected site in the anti-sense stand includes an A(or a modified base which pairs with T), the corresponding monomer inthe target is a T, and the sense strand is chosen from a base which willnot pair or which will form a noncanonical pair, e.g., G;

the monomer at the selected site in the anti-sense stand includes a U(or a modified base which pairs with A), the corresponding monomer inthe target is an A, and the sense strand is chosen from a monomer whichwill not pair or which will form a non-canonical pairing, e.g., U or G;

the monomer at the selected site in the anti-sense stand includes a C(or a modified base which pairs with G), the corresponding monomer inthe target is a G, and the sense strand is chosen a monomer which willnot pair or which will form a non-canonical pairing, e.g., G,A_(cis)/A_(trans), or U; or

the monomer at the selected site in the anti-sense stand includes a G(or a modified base which pairs with C), the corresponding monomer inthe target is a C, and the sense strand is chosen from a monomer whichwill not pair or which will form a non-canonical pairing.

In another embodiment a non-naturally occurring or modified monomer ormonomers is chosen such that when it occupies complementary a positionin an iRNA agent they exhibit a first free energy of dissociation andwhen one (or both) of them pairs with a naturally occurring monomer, thepair exhibits a second free energy of dissociation, which is usuallyhigher than that of the pairing of the first and second monomers. E.g.,when the first and second monomers occupy complementary positions theyeither do not pair and have no substantial level of H-bonding, or form aweaker bond than one of them would form with a naturally occurringmonomer, and reduce the stability of that duplex, but when the duplexdissociates at least one of the strands will form a duplex with a targetin which the selected monomer will promote stability, e.g., the monomerwill form a more stable pair with a naturally occurring monomer in thetarget sequence than the pairing it formed in the iRNA agent.

An example of such a pairing is 2-amino A and either of a 2-thiopyrimidine analog of U or T. As is discussed above, when placed incomplementary positions of the iRNA agent these monomers will pair verypoorly and will minimize stability. However, a duplex is formed between2 amino A and the U of a naturally occurring target, or a duplex isformed between 2-thio U and the A of a naturally occurring target or2-thio T and the A of a naturally occurring target will have arelatively higher free energy of dissociation and be more stable.

The monomer at the selected position in the sense strand can be auniversal pairing moiety. A universal pairing agent will form some levelof H bonding with more than one and preferably all other naturallyoccurring monomers. An examples of a universal pairing moiety is amonomer which includes 3-nitro pyrrole. Examples of other candidateuniversal base analogs can be found in the art, e.g., in Loakes, 2001,NAR 29: 2437-2447, hereby incorporated by reference. In these cases themonomer at the corresponding position of the anti-sense strand can bechosen for its ability to form a duplex with the target and can include,e.g., A, U, G, or C.

In another aspect, the invention features, an iRNA agent which includes:

a sense sequence, which preferably does not target a sequence in asubject, and an anti-sense sequence, which targets a plurality of targetsequences in a subject, wherein the targets differ in sequence at only 1or a small number, e.g., no more than 5, 4, 3 or 2 positions. The senseand anti-sense sequences have sufficient complementarity to each otherto hybridize, e.g., under physiological conditions, e.g., underphysiological conditions but not in contact with a helicase or otherunwinding enzyme. In the sequence of the anti-sense strand of the iRNAagent is selected such that at one, some, or all of the positions whichcorrespond to positions that differe in sequence between the targetsequences, the anti-sense strand will include a monomer which will formH-bonds with at least two different target sequences. In a preferredexample the anti-sense sequence will include a universal or promiscuousmonomer, e.g., a monomer which includes 5-nitro pyrrole, 2-amino A,2-thio U or 2-thio T, or other universal base referred to herein.

In a preferred embodiment the iRNA agent targets repeated sequences(which differ at only one or a small number of positions from eachother) in a single gene, a plurality of genes, or a viral genome, e.g.,the HCV genome.

An embodiment is illustrated in the FIGS. 13 and 14.

In another aspect, the invention features, determining, e.g., bymeasurement or calculation, the stability of a pairing between monomersat a selected or constrained position in the iRNA agent duplex, andpreferably determining the stability for the corresponding pairing in aduplex between a sequence form the iRNA agent and another RNA, e.g., atarget sequence. The determinations can be compared. An iRNA agent thusanalysed can be used in the development of a further modified iRNA agentor can be administered to a subject. This analysis can be performedsuccessively to refine or desing optimized iRNA agents.

In another aspect, the invention features, a kit which includes one ormore of the following an iRNA described herein, a sterile container inwhich the iRNA agent is disclosed, and instructions for use.

In another aspect, the invention features, an iRNA agent containing aconstrained sequence made by a method described herein. The iRNA agentcan target one or more of the genes referred to herein.

iRNA agents having constrained or selected sites, e.g., as describedherein, can be used in any way described herein. Accordingly, they iRNAagents having constrained or selected sites, e.g., as described herein,can be used to silence a target, e.g., in any of the methods describedherein and to target any of the genes described herein or to treat anyof the disorders described herein. iRNA agents having constrained orselected sites, e.g., as described herein, can be incorporated into anyof the formulations or preparations, e.g., pharmaceutical or sterilepreparations described herein. iRNA agents having constrained orselected sites, e.g., as described herein, can be administered by any ofthe routes of administration described herein.

The term “other than canonical Watson-Crick pairing” as used herein,refers to a pairing between a first monomer in a first sequence and asecond monomer at the corresponding position in a second sequence of aduplex in which one or more of the following is true: (1) there isessentially no pairing between the two, e.g., there is no significantlevel of H bonding between the monomers or binding between the monomersdoes not contribute in any significant way to the stability of theduplex; (2) the monomers are a non-canonical paring of monomers having anaturally occurring bases, i.e., they are other than A-T, A-U, or G-C,and they form monomer-monomer H bonds, although generally the H bondingpattern formed is less strong than the bonds formed by a canonicalpairing; or (3) at least one of the monomers includes a non-naturallyoccurring bases and the H bonds formed between the monomers is,preferably formed is less strong than the bonds formed by a canonicalpairing, namely one or more of A-T, A-U, G-C.

The term “off-target” as used herein, refers to as a sequence other thanthe sequence to be silenced.

Universal Bases: “wild-cards”; shape-based complementarity

Bi-stranded, multisite replication of a base pair betweendifluorotoluene and adenine: confirmation by ‘inverse’ sequencing. Liu,D.; Moran, S.; Kool, E. T. Chem. Biol., 1997, 4, 919-926)

(Importance of terminal base pair hydrogen-bonding in 3′-endproofreading by the Klenow fragment of DNA polymerase I. Morales, J. C.;Kool, E. T. Biochemistry, 2000, 39, 2626-2632)

(Selective and stable DNA base pairing without hydrogen bonds. Matray,T, J.; Kool, E. T. J. Am. Chem. Soc., 1998, 120, 6191-6192)

(Difluorotoluene, a nonpolar isostere for thymine, codes specificallyand efficiently for adenine in DNA replication. Moran, S. Ren, R. X.-F.;Rumney I V, S.; Kool, E. T. J. Am. Chem. Soc., 1997, 119, 2056-2057)

(Structure and base pairing properties of a replicable nonpolar isosterefor deoxyadenosine. Guckian, K. M.; Morales, J. C.; Kool, E. T. J. Org.Chem., 1998, 63, 9652-9656)

(Universal bases for hybridization, replication and chain termination.Berger, M.; Wu. Y.; Ogawa, A. K.;

McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. Nucleic Acids Res.,2000, 28, 2911-2914)

-   (1. Efforts toward the expansion of the genetic alphabet:    Information storage and replication with unnatural hydrophobic base    pairs. Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.;    Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 3274-3287. 2.    Rational design of an unnatural base pair with increased kinetic    selectivity. Ogawa, A. K.; Wu. Y.; Berger, M.; Schultz, P. G.;    Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 8803-8804)

(Efforts toward expansion of the genetic alphabet: replication of DNAwith three base pairs. Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.;Romesberg, F. E. J. Am. Chem. Soc., 2001, 123, 7439-7440)

(1. Efforts toward expansion of the genetic alphabet: Optimization ofinterbase hydrophobic interactions. Wu, Y.; Ogawa, A. K.; Berger, M.;McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000,122, 7621-7632. 2. Efforts toward expansion of genetic alphabet: DNApolymerase recognition of a highly stable, self-pairing hydrophobicbase. McMinn, D. L.; Ogawa. A. K.; Wu, Y.; Liu, J.; Schultz, P. G.;Romesberg, F. E. J. Am. Chem. Soc., 1999, 121, 11585-11586)

(A stable DNA duplex containing a non-hydrogen-bonding and non-shapecomplementary base couple: Interstrand stacking as the stabilitydetermining factor. Brotschi, C.; Haberli, A.; Leumann, C, J. Angew.Chem. Int. Ed., 2001, 40, 3012-3014)

(2,2′-Bipyridine Ligandoside: A novel building block for modifying DNAwith intra-duplex metal complexes. Weizman, H.; Tor, Y. J. Am. Chem.Soc., 2001, 123, 3375-3376)

(Minor groove hydration is critical to the stability of DNA duplexes.Lan, T.; McLaughlin, L. W. J. Am. Chem. Soc., 2000, 122, 6512-13)

(Effect of the Universal base 3-nitropyrrole on the selectivity ofneighboring natural bases. Oliver, J. S.; Parker, K. A.; Suggs, J. W.Organic Lett., 2001, 3, 1977-1980. 2. Effect of the1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrol residue on the stability ofDNA duplexes and triplexes. Amosova, O.; George J.; Fresco, J. R.Nucleic Acids Res., 1997, 25, 1930-1934. 3. Synthesis, structure anddeoxyribonucleic acid sequencing with a universal nucleosides:1-(2′-deoxy-(3-D-ribofuranosyl)-3-nitropyrrole. Bergstrom, D. E.; Zhang,P.; Toma, P. H.; Andrews, P. C.; Nichols, R. J. Am. Chem. Soc., 1995,117, 1201-1209) (

(Model studies directed toward a general triplex DNA recognition scheme:a novel DNA base that binds a CG base-pair in an organic solvent.Zimmerman, S. C.; Schmitt, P. J. Am. Chem. Soc., 1995, 117, 10769-10770)

(A universal, photocleavable DNA base: nitropiperonyl 2′-deoxyriboside.J. Org. Chem., 2001, 66, 2067-2071)

(Recognition of a single guanine bulge by 2-acylamino-1,8-naphthyridine.Nakatani, K.; Sando, S.; Saito, I. J. Am. Chem. Soc., 2000, 122,2172-2177. b. Specific binding of 2-amino-1,8-naphthyridine into singleguanine bulge as evidenced by photooxidation of GC doublet, Nakatani,K.; Sando, S.; Yoshida, K.; Saito, I. Bioorg. Med. Chem. Lett., 2001,11, 335-337)

Asymmetrical Modifications

The monomers and methods described herein can be used to prepare an RNA,e.g., an iRNA agent, that can be asymmetrically modified as describedherein, and as described in International Application Serial No.PCT/US04/07070, filed Mar. 8, 2004, which is hereby incorporated byreference.

An asymmetrically modified iRNA agent is one in which a strand has amodification which is not present on the other strand. An asymmetricalmodification is a modification found on one strand but not on the otherstrand. Any modification, e.g., any modification described herein, canbe present as an asymmetrical modification. An asymmetrical modificationcan confer any of the desired properties associated with a modification,e.g., those properties discussed herein. E.g., an asymmetricalmodification can: confer resistance to degradation, an alteration inhalf life; target the iRNA agent to a particular target, e.g., to aparticular tissue; modulate, e.g., increase or decrease, the affinity ofa strand for its complement or target sequence; or hinder or promotemodification of a terminal moiety, e.g., modification by a kinase orother enzymes involved in the RISC mechanism pathway. The designation ofa modification as having one property does not mean that it has no otherproperty, e.g., a modification referred to as one which promotesstabilization might also enhance targeting.

While not wishing to be bound by theory or any particular mechanisticmodel, it is believed that asymmetrical modification allows an iRNAagent to be optimized in view of the different or “asymmetrical”functions of the sense and antisense strands. For example, both strandscan be modified to increase nuclease resistance, however, since somechanges can inhibit RISC activity, these changes can be chosen for thesense stand. In addition, since some modifications, e.g., targetingmoieties, can add large bulky groups that, e.g., can interfere with thecleavage activity of the RISC complex, such modifications are preferablyplaced on the sense strand. Thus, targeting moieties, especially bulkyones (e.g. cholesterol), are preferentially added to the sense strand.In one embodiment, an asymmetrical modification in which a phosphate ofthe backbone is substituted with S, e.g., a phosphorothioatemodification, is present in the antisense strand, and a 2′ modification,e.g., 2′ OMe is present in the sense strand. A targeting moiety can bepresent at either (or both) the 5′ or 3′ end of the sense strand of theiRNA agent. In a preferred example, a P of the backbone is replaced withS in the antisense strand, 2′OMe is present in the sense strand, and atargeting moiety is added to either the 5′ or 3′ end of the sense strandof the iRNA agent.

In a preferred embodiment an asymmetrically modified iRNA agent has amodification on the sense strand which modification is not found on theantisense strand and the antisense strand has a modification which isnot found on the sense strand.

Each strand can include one or more asymmetrical modifications. By wayof example: one strand can include a first asymmetrical modificationwhich confers a first property on the iRNA agent and the other strandcan have a second asymmetrical modification which confers a secondproperty on the iRNA. E.g., one strand, e.g., the sense strand can havea modification which targets the iRNA agent to a tissue, and the otherstrand, e.g., the antisense strand, has a modification which promoteshybridization with the target gene sequence.

In some embodiments both strands can be modified to optimize the sameproperty, e.g., to increase resistance to nucleolytic degradation, butdifferent modifications are chosen for the sense and the antisensestrands, e.g., because the modifications affect other properties aswell. E.g., since some changes can affect RISC activity thesemodifications are chosen for the sense strand.

In an embodiment one strand has an asymmetrical 2′ modification, e.g., a2′ OMe modification, and the other strand has an asymmetricalmodification of the phosphate backbone, e.g., a phosphorothioatemodification. So, in one embodiment the antisense strand has anasymmetrical 2′ OMe modification and the sense strand has anasymmetrical phosphorothioate modification (or vice versa). In aparticularly preferred embodiment the RNAi agent will have asymmetrical2′-O alkyl, preferably, 2′-OMe modifications on the sense strand andasymmetrical backbone P modification, preferably a phosphothioatemodification in the antisense strand. There can be one or multiple2′-OMe modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunitsof the sense strand can be so modified. There can be one or multiplephosphorothioate modifications, e.g., at least 2, 3, 4, 5, or 6, of thesubunits of the antisense strand can be so modified. It is preferable tohave an iRNA agent wherein there are multiple 2′-OMe modifications onthe sense strand and multiple phosphorothioate modifications on theantisense strand. All of the subunits on one or both strands can be somodified. A particularly preferred embodiment of multiple asymmetricmodification on both strands has a duplex region about 20-21, andpreferably 19, subunits in length and one or two 3′ overhangs of about 2subunits in length.

Asymmetrical modifications are useful for promoting resistance todegradation by nucleases, e.g., endonucleases. iRNA agents can includeone or more asymmetrical modifications which promote resistance todegradation. In preferred embodiments the modification on the antisensestrand is one which will not interfere with silencing of the target,e.g., one which will not interfere with cleavage of the target. Most ifnot all sites on a strand are vulnerable, to some degree, to degradationby endonucleases. One can determine sites which are relativelyvulnerable and insert asymmetrical modifications which inhibitdegradation. It is often desirable to provide asymmetrical modificationof a UA site in an iRNA agent, and in some cases it is desirable toprovide the UA sequence on both strands with asymmetrical modification.Examples of modifications which inhibit endonucleolytic degradation canbe found herein. Particularly favored modifications include: 2′modification, e.g., provision of a 2′ OMe moiety on the U, especially ona sense strand; modification of the backbone, e.g., with the replacementof an O with an S, in the phosphate backbone, e.g., the provision of aphosphorothioate modification, on the U or the A or both, especially onan antisense strand; replacement of the U with a C5 amino linker;replacement of the A with a G (sequence changes are preferred to belocated on the sense strand and not the antisense strand); andmodification of the at the 2′, 6′, 7′, or 8′ position. Preferredembodiments are those in which one or more of these modifications arepresent on the sense but not the antisense strand, or embodiments wherethe antisense strand has fewer of such modifications.

Asymmetrical modification can be used to inhibit degradation byexonucleases. Asymmetrical modifications can include those in which onlyone strand is modified as well as those in which both are modified. Inpreferred embodiments the modification on the antisense strand is onewhich will not interfere with silencing of the target, e.g., one whichwill not interfere with cleavage of the target. Some embodiments willhave an asymmetrical modification on the sense strand, e.g., in a 3′overhang, e.g., at the 3′ terminus, and on the antisense strand, e.g.,in a 3′ overhang, e.g., at the 3′ terminus. If the modificationsintroduce moieties of different size it is preferable that the larger beon the sense strand. If the modifications introduce moieties ofdifferent charge it is preferable that the one with greater charge be onthe sense strand.

Examples of modifications which inhibit exonucleolytic degradation canbe found herein. Particularly favored modifications include: 2′modification, e.g., provision of a 2′ OMe moiety in a 3′ overhang, e.g.,at the 3′ terminus (3′ terminus means at the 3′ atom of the molecule orat the most 3′ moiety, e.g., the most 3′ P or 2′ position, as indicatedby the context); modification of the backbone, e.g., with thereplacement of a P with an S, e.g., the provision of a phosphorothioatemodification, or the use of a methylated P in a 3′ overhang, e.g., atthe 3′ terminus; combination of a 2′ modification, e.g., provision of a2′ 0 Me moiety and modification of the backbone, e.g., with thereplacement of a P with an S, e.g., the provision of a phosphorothioatemodification, or the use of a methylated P, in a 3′ overhang, e.g., atthe 3′ terminus; modification with a 3′ alkyl; modification with anabasic pyrrolidine in a 3′ overhang, e.g., at the 3′ terminus;modification with naproxene, ibuprofen, or other moieties which inhibitdegradation at the 3′ terminus. Preferred embodiments are those in whichone or more of these modifications are present on the sense but not theantisense strand, or embodiments where the antisense strand has fewer ofsuch modifications.

Modifications, e.g., those described herein, which affect targeting canbe provided as asymmetrical modifications. Targeting modifications whichcan inhibit silencing, e.g., by inhibiting cleavage of a target, can beprovided as asymmetrical modifications of the sense strand. Abiodistribution altering moiety, e.g., cholesterol, can be provided inone or more, e.g., two, asymmetrical modifications of the sense strand.Targeting modifications which introduce moieties having a relativelylarge molecular weight, e.g., a molecular weight of more than 400, 500,or 1000 daltons, or which introduce a charged moiety (e.g., having morethan one positive charge or one negative charge) can be placed on thesense strand.

Modifications, e.g., those described herein, which modulate, e.g.,increase or decrease, the affinity of a strand for its compliment ortarget, can be provided as asymmetrical modifications. These include: 5methyl U; 5 methyl C; pseudouridine, Locked nucleic acids,2 thio U and2-amino-A. In some embodiments one or more of these is provided on theantisense strand.

iRNA agents have a defined structure, with a sense strand and anantisense strand, and in many cases short single strand overhangs, e.g.,of 2 or 3 nucleotides are present at one or both 3′ ends. Asymmetricalmodification can be used to optimize the activity of such a structure,e.g., by being placed selectively within the iRNA. E.g., the end regionof the iRNA agent defined by the 5′ end of the sense strand and the 3′end of the antisense strand is important for function. This region caninclude the terminal 2, 3, or 4 paired nucleotides and any 3′ overhang.In preferred embodiments asymmetrical modifications which result in oneor more of the following are used: modifications of the 5′ end of thesense strand which inhibit kinase activation of the sense strand,including, e.g., attachments of conjugates which target the molecule orthe use modifications which protect against 5′ exonucleolyticdegradation; or modifications of either strand, but preferably the sensestrand, which enhance binding between the sense and antisense strand andthereby promote a “tight” structure at this end of the molecule.

The end region of the iRNA agent defined by the 3′ end of the sensestrand and the 5′ end of the antisense strand is also important forfunction. This region can include the terminal 2, 3, or 4 pairednucleotides and any 3′ overhang. Preferred embodiments includeasymmetrical modifications of either strand, but preferably the sensestrand, which decrease binding between the sense and antisense strandand thereby promote an “open” structure at this end of the molecule.Such modifications include placing conjugates which target the moleculeor modifications which promote nuclease resistance on the sense strandin this region. Modification of the antisense strand which inhibitkinase activation are avoided in preferred embodiments.

Exemplary modifications for asymmetrical placement in the sense strandinclude the following:

(a) backbone modifications, e.g., modification of a backbone P,including replacement of P with S, or P substituted with alkyl or allyl,e.g., Me, and dithioates (S—P═S); these modifications can be used topromote nuclease resistance;

(b) 2′-O alkyl, e.g., 2′-OMe, 3′-O alkyl, e.g., 3′-OMe (at terminaland/or internal positions); these modifications can be used to promotenuclease resistance or to enhance binding of the sense to the antisensestrand, the 3′ modifications can be used at the 5′ end of the sensestrand to avoid sense strand activation by RISC;

(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe and with P═O or P═S)these modifications can be used to promote nuclease resistance or toinhibit binding of the sense to the antisense strand, or can be used atthe 5′ end of the sense strand to avoid sense strand activation by RISC;

(d) L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe);these modifications can be used to promote nuclease resistance or toinhibit binding of the sense to the antisense strand, or can be used atthe 5′ end of the sense strand to avoid sense strand activation by RISC;

(e) modified sugars (e.g., locked nucleic acids (LNA's), hexose nucleicacids (HNA's) and cyclohexene nucleic acids (CeNA's)); thesemodifications can be used to promote nuclease resistance or to inhibitbinding of the sense to the antisense strand, or can be used at the 5′end of the sense strand to avoid sense strand activation by RISC;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2modified purines, N-7 modified purines, N-6 modified purines), thesemodifications can be used to promote nuclease resistance or to enhancebinding of the sense to the antisense strand;

(g) cationic groups and Zwitterionic groups (preferably at a terminus),these modifications can be used to promote nuclease resistance;

(h) conjugate groups (preferably at terminal positions), e,g., naproxen,biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates;these modifications can be used to promote nuclease resistance or totarget the molecule, or can be used at the 5′ end of the sense strand toavoid sense strand activation by RISC.

Exemplary modifications for asymmetrical placement in the antisensestrand include the following:

(a) backbone modifications, e.g., modification of a backbone P,including replacement of P with S, or P substituted with alkyl or allyl,e.g., Me, and dithioates (S—P═S);

(b) 2′-O alkyl, e.g., 2′-OMe, (at terminal positions);

(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe) e.g., terminal at the3′ end); e.g., with P═O or P═S preferably at the 3′-end, thesemodifications are preferably excluded from the 5′ end region as they mayinterfere with RISC enzyme activity such as kinase activity;

(d) L sugars (e.g, L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe);e.g., terminal at the 3′ end; e.g., with P═O or P═S preferably at the3′-end, these modifications are preferably excluded from the 5′ endregion as they may interfere with kinase activity;

(e) modified sugars (e.g., LNA's, HNA's and CeNA's); these modificationsare preferably excluded from the 5′ end region as they may contribute tounwanted enhancements of paring between the sense and antisense strands,it is often preferred to have a “loose” structure in the 5′ region,additionally, they may interfere with kinase activity;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2modified purines, N-7 modified purines, N-6 modified purines);

(g) cationic groups and Zwitterionic groups (preferably at a terminus);conjugate groups (preferably at terminal positions), e,g., naproxen,biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates,but bulky groups or generally groups which inhibit RISC activity shouldare less preferred.

The 5′-OH of the antisense strand should be kept free to promoteactivity. In some preferred embodiments modifications that promotenuclease resistance should be included at the 3′ end, particularly inthe 3′ overhang.

In another aspect, the invention features a method of optimizing, e.g.,stabilizing, an iRNA agent. The method includes selecting a sequencehaving activity, introducing one or more asymmetric modifications intothe sequence, wherein the introduction of the asymmetric modificationoptimizes a property of the iRNA agent but does not result in a decreasein activity.

The decrease in activity can be less than a preselected level ofdecrease. In preferred embodiments decrease in activity means a decreaseof less than 5, 10, 20, 40, or 50% activity, as compared with anotherwise similar iRNA lacking the introduced modification. Activitycan, e.g., be measured in vivo, or in vitro, with a result in eitherbeing sufficient to demonstrate the required maintenance of activity.

The optimized property can be any property described herein and inparticular the properties discussed in the section on asymmetricalmodifications provided herein. The modification can be any asymmetricalmodification, e.g., an asymmetric modification described in the sectionon asymmetrical modifications described herein. Particularly preferredasymmetric modifications are 2′-O alkyl modifications, e.g., 2′-OMemodifications, particularly in the sense sequence, and modifications ofa backbone O, particularly phosphorothioate modifications, in theantisense sequence.

In a preferred embodiment a sense sequence is selected and provided withan asymmetrical modification, while in other embodiments an antisensesequence is selected and provided with an asymmetrical modification. Insome embodiments both sense and antisense sequences are selected andeach provided with one or more asymmetrical modifications.

Multiple asymmetric modifications can be introduced into either or bothof the sense and antisense sequence. A sequence can have at least 2, 4,6, 8, or more modifications and all or substantially all of the monomersof a sequence can be modified.

Table 2 shows examples having strand I with a selected modification andstrand II with a selected modification.

TABLE 2 Exemplary strand I- and strand II-modifications Strand I StrandII Nuclease Resistance (e.g., 2′-OMe) Biodistribution (e.g., P═S)Biodistribution conjugate Protein Binding Functionality (e.g.,Lipophile) (e.g., Naproxen) Tissue Distribution Functionality CellTargeting Functionality (e.g., Carbohydrates) (e.g., Folate for cancercells) Tissue Distribution Functionality Fusogenic Functionality (e.g.,Kidney Cell Targetingmoieties) (e.g., Polyethylene imines) Cancer CellTargeting Fusogenic Functionality (e.g., RGD peptides and imines) (e.g.,peptides) Nuclease Resistance (e.g., 2′-OMe) Increase in bindingAffinity (5-Me—C, 5-Me—U, 2-thio-U, 2-amino-A, G-clamp, LNA) TissueDistribution Functionality RISC activity improving Functionality Helicalconformation changing Tissue Distribution Functionality Functionalities(P═S; lipophile, carbohydrates)

Z—X—Y Architecture

The monomers and methods described herein can be used to prepare an RNA,e.g., an iRNA agent, having a Z—X—Y architecture or structure such asthose described herein and those described in copending, co-owned U.S.Provisional Application Ser. No. 60/510,246, filed on Oct. 9, 2003,which is hereby incorporated by reference, copending, co-owned U.S.Provisional Application Ser. No. 60/510,318, filed on Oct. 10, 2003,which is hereby incorporated by reference, and copending, co-ownedInternational Application No. PCT/US04/07070, filed Mar. 8, 2004.

Thus, an iRNA agent can have a first segment, the Z region, a secondsegment, the X region, and optionally a third region, the Y region:

Z—X—Y.

It may be desirable to modify subunits in one or both of Zand/or Y onone hand and X on the other hand. In some cases they will have the samemodification or the same class of modification but it will more often bethe case that the modifications made in Z and/or Y will differ fromthose made in X.

The Z region typically includes a terminus of an iRNA agent. The lengthof the Z region can vary, but will typically be from 2-14, morepreferably 2-10, subunits in length. It typically is single stranded,i.e., it will not base pair with bases of another strand, though it mayin some embodiments self associate, e.g., to form a loop structure. Suchstructures can be formed by the end of a strand looping back and formingan intrastrand duplex. E.g., 2, 3, 4, 5 or more intra-strand bases pairscan form, having a looped out or connecting region, typically of 2 ormore subunits which do not pair. This can occur at one or both ends of astrand. A typical embodiment of a Z region is a single strand overhang,e.g., an over hang of the length described elsewhere herein. The Zregion can thus be or include a 3′ or 5′ terminal single strand. It canbe sense or antisense strand but if it is antisense it is preferred thatit is a 3-overhang. Typical inter-subunit bonds in the Z region include:P═O; P═S; S—P═S; P—NR₂; and P—BR₂. Chiral P═X, where X is S, N, or B)inter-subunit bonds can also be present. (These inter-subunit bonds arediscussed in more detail elsewhere herein.) Other preferred Z regionsubunit modifications (also discussed elsewhere herein) can include:3′-OR, 3′SR, 2′-OMe, 3′-OMe, and 2′OH modifications and moieties; alphaconfiguration bases; and 2′ arabino modifications.

The X region will in most cases be duplexed, in the case of a singlestrand iRNA agent, with a corresponding region of the single strand, orin the case of a double stranded iRNA agent, with the correspondingregion of the other strand. The length of the X region can vary but willtypically be between 10-45 and more preferably between 15 and 35subunits. Particularly preferred region X's will include 17, 18, 19, 29,21, 22, 23, 24, or 25 nucleotide pairs, though other suitable lengthsare described elsewhere herein and can be used. Typical X regionsubunits include 2′-OH subunits. In typical embodiments phosphateinter-subunit bonds are preferred while phosphorothioate ornon-phosphate bonds are absent. Other modifications preferred in the Xregion include: modifications to improve binding, e.g., nucleobasemodifications; cationic nucleobase modifications; and C-5 modifiedpyrimidines, e.g., allylamines. Some embodiments have 4 or moreconsecutive 2′OH subunits. While the use of phosphorothioate issometimes non preferred they can be used if they connect less than 4consecutive 2′OH subunits.

The Y region will generally conform to the parameters set out for the Zregions. However, the X and Z regions need not be the same, differenttypes and numbers of modifications can be present, and infact, one willusually be a 3′ overhang and one will usually be a 5′ overhang.

In a preferred embodiment the iRNA agent will have a Y and/or Z regioneach having ribonucleosides in which the 2′-OH is substituted, e.g.,with 2′-OMe or other alkyl; and an X region that includes at least fourconsecutive ribonucleoside subunits in which the 2′-OH remainsunsubstituted.

The subunit linkages (the linkages between subunits) of an iRNA agentcan be modified, e.g., to promote resistance to degradation. Numerousexamples of such modifications are disclosed herein, one example ofwhich is the phosphorothioate linkage. These modifications can beprovided between the subunits of any of the regions, Y, X, and Z.However, it is preferred that their occurrences minimized and inparticular it is preferred that consecutive modified linkages beavoided.

In a preferred embodiment the iRNA agent will have a Y and Z region eachhaving ribonucleosides in which the 2′-OH is substituted, e.g., with2′-OMe; and an X region that includes at least four consecutivesubunits, e.g., ribonucleoside subunits in which the 2′-OH remainsunsubstituted.

As mentioned above, the subunit linkages of an iRNA agent can bemodified, e.g., to promote resistance to degradation. Thesemodifications can be provided between the subunits of any of theregions, Y, X, and Z. However, it is preferred that they are minimizedand in particular it is preferred that consecutive modified linkages beavoided.

Thus, in a preferred embodiment, not all of the subunit linkages of theiRNA agent are modified and more preferably the maximum number ofconsecutive subunits linked by other than a phosphodiester bond will be2, 3, or 4. Particularly preferred iRNA agents will not have four ormore consecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, inwhich each subunits is joined by modified linkages—i.e. linkages thathave been modified to stabilize them from degradation as compared to thephosphodiester linkages that naturally occur in RNA and DNA.

It is particularly preferred to minimize the occurrence in region X.Thus, in preferred embodiments each of the nucleoside subunit linkagesin X will be phosphodiester linkages, or if subunit linkages in region Xare modified, such modifications will be minimized. E.g., although the Yand/or Z regions can include inter subunit linkages which have beenstabilized against degradation, such modifications will be minimized inthe X region, and in particular consecutive modifications will beminimized. Thus, in preferred embodiments the maximum number ofconsecutive subunits linked by other than a phosphodiester bond will be2, 3, or 4. Particularly preferred X regions will not have four or moreconsecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, inwhich each subunits is joined by modified linkages—i.e. linkages thathave been modified to stabilize them from degradation as compared to thephosphodiester linkages that naturally occur in RNA and DNA.

In a preferred embodiment Y and/or Z will be free of phosphorothioatelinkages, though either or both may contain other modifications, e.g.,other modifications of the subunit linkages.

In a preferred embodiment region X, or in some cases, the entire iRNAagent, has no more than 3 or no more than 4 subunits having identical 2′moieties.

In a preferred embodiment region X, or in some cases, the entire iRNAagent, has no more than 3 or no more than 4 subunits having identicalsubunit linkages.

In a preferred embodiment one or more phosphorothioate linkages (orother modifications of the subunit linkage) are present in Y and/or Z,but such modified linkages do not connect two adjacent subunits, e.g.,nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g.,any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by alinkage other than a a phosphorothioate linkage.

In a preferred embodiment each of Y and/or Z independently has only onephosphorothioate linkage between adjacent subunits, e.g., nucleosides,having a 2′ modification, e.g., 2′-O-alkyl nucleosides. If there is asecond set of adjacent subunits, e.g., nucleosides, having a 2′modification, e.g., 2′-O-alkyl nucleosides, in Y and/or Z that secondset is connected by a linkage other than a phosphorothioate linkage,e.g., a modified linkage other than a phosphorothioate linkage.

In a preferred embodiment each of Y and/or Z independently has more thanone phosphorothioate linkage connecting adjacent pairs of subunits,e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkylnucleosides, but at least one pair of adjacent subunits, e.g.,nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, arebe connected by a linkage other than a phosphorothioate linkage, e.g., amodified linkage other than a phosphorothioate linkage.

In a preferred embodiment one of the above recited limitation onadjacent subunits in Y and or Z is combined with a limitation on thesubunits in X. E.g., one or more phosphorothioate linkages (or othermodifications of the subunit linkage) are present in Y and/or Z, butsuch modified linkages do not connect two adjacent subunits, e.g.,nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g.,any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by alinkage other than a phosphorothioate linkage. In addition, the X regionhas no more than 3 or no more than 4 identical subunits, e.g., subunitshaving identical 2′ moieties or the X region has no more than 3 or nomore than 4 subunits having identical subunit linkages.

A Y and/or Z region can include at least one, and preferably 2, 3 or 4of a modification disclosed herein. Such modifications can be chosen,independently, from any modification described herein, e.g., fromnuclease resistant subunits, subunits with modified bases, subunits withmodified intersubunit linkages, subunits with modified sugars, andsubunits linked to another moiety, e.g., a targeting moiety. In apreferred embodiment more than 1 of such subunits can be present but insome embodiments it is preferred that no more than 1, 2, 3, or 4 of suchmodifications occur, or occur consecutively. In a preferred embodimentthe frequency of the modification will differ between Y and/or Z and X,e.g., the modification will be present one of Y and/or Z or X and absentin the other.

An X region can include at least one, and preferably 2, 3 or 4 of amodification disclosed herein. Such modifications can be chosen,independently, from any modification described herein, e.g., fromnuclease resistant subunits, subunits with modified bases, subunits withmodified intersubunit linkages, subunits with modified sugars, andsubunits linked to another moiety, e.g., a targeting moiety. In apreferred embodiment more than 1 of such subunits can b present but insome embodiments it is preferred that no more than 1, 2, 3, or 4 of suchmodifications occur, or occur consecutively.

An RRMS (described elsewhere herein) can be introduced at one or morepoints in one or both strands of a double-stranded iRNA agent. An RRMScan be placed in a Y and/or Z region, at or near (within 1, 2, or 3positions) of the 3′ or 5′ end of the sense strand or at near (within 2or 3 positions of) the 3′ end of the antisense strand. In someembodiments it is preferred to not have an RRMS at or near (within 1, 2,or 3 positions of) the 5′ end of the antisense strand. An RRMS can bepositioned in the X region, and will preferably be positioned in thesense strand or in an area of the antisense strand not critical forantisense binding to the target.

Differential Modification of Terminal Duplex Stability

In one aspect, the monomers and methods described herein can be used toprepare an iRNA agent having differential modification of terminalduplex stability (DMTDS).

In addition, the monomers and methods described herein can be used toprepare iRNA agents having DMTDS and another element described herein.E.g., the monomers and methods described herein can be used to preparean iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNAagent having a non canonical pairing, an iRNA agent which targets a genedescribed herein, e.g., a gene active in the kidney, an iRNA agenthaving an architecture or structure described herein, an iRNA associatedwith an amphipathic delivery agent described herein, an iRNA associatedwith a drug delivery module described herein, an iRNA agent administeredas described herein, or an iRNA agent formulated as described herein,which also incorporates DMTDS.

iRNA agents can be optimized by increasing the propensity of the duplexto disassociate or melt (decreasing the free energy of duplexassociation), in the region of the 5′ end of the antisense strandduplex. This can be accomplished, e.g., by the inclusion of subunitswhich increase the propensity of the duplex to disassociate or melt inthe region of the 5′ end of the antisense strand. It can also beaccomplished by the attachment of a ligand that increases the propensityof the duplex to disassociate of melt in the region of the 5′ end. Whilenot wishing to be bound by theory, the effect may be due to promotingthe effect of an enzyme such as helicase, for example, promoting theeffect of the enzyme in the proximity of the 5′ end of the antisensestrand.

The inventors have also discovered that iRNA agents can be optimized bydecreasing the propensity of the duplex to disassociate or melt(increasing the free energy of duplex association), in the region of the3′ end of the antisense strand duplex. This can be accomplished, e.g.,by the inclusion of subunits which decrease the propensity of the duplexto disassociate or melt in the region of the 3′ end of the antisensestrand. It can also be accomplished by the attachment of ligand thatdecreases the propensity of the duplex to disassociate of melt in theregion of the 5′ end.

Modifications which increase the tendency of the 5′ end of the duplex todissociate can be used alone or in combination with other modificationsdescribed herein, e.g., with modifications which decrease the tendencyof the 3′ end of the duplex to dissociate. Likewise, modifications whichdecrease the tendency of the 3′ end of the duplex to dissociate can beused alone or in combination with other modifications described herein,e.g., with modifications which increase the tendency of the 5′ end ofthe duplex to dissociate.

Decreasing the Stability of the AS 5′ End of the Duplex

Subunit pairs can be ranked on the basis of their propensity to promotedissociation or melting (e.g., on the free energy of association ordissociation of a particular pairing, the simplest approach is toexamine the pairs on an individual pair basis, though next neighbor orsimilar analysis can also be used). In terms of promoting dissociation:

-   -   A:U is preferred over G:C;    -   G:U is preferred over G:C;    -   I:C is preferred over G:C (I=inosine);

mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings;

pairings which include a universal base are preferred over canonicalpairings.

A typical ds iRNA agent can be diagrammed as follows:

S 5' R₁ N₁ N₂ N₃ N₄ N₅ [N] N⁻⁵ N⁻⁴ N⁻³ N⁻² N⁻¹ R₂ 3' AS 3' R₃ N₁ N₂ N₃N₄ N₅ [N] N⁻⁵ N⁻⁴ N⁻³ N⁻² N⁻¹ R₄ 5' S:AS P₁ P₂ P₃ P₄ P₅ [N] P⁻⁵ P⁻⁴ P⁻³P⁻² P⁻¹ 5'

S indicates the sense strand; AS indicates antisense strand; R₁indicates an optional (and nonpreferred) 5′ sense strand overhang; R₂indicates an optional (though preferred) 3′ sense overhang; R₃ indicatesan optional (though preferred) 3′ antisense sense overhang; R₄ indicatesan optional (and nonpreferred) 5′ antisense overhang; N indicatessubunits; [N] indicates that additional subunit pairs may be present;and P_(X), indicates a paring of sense N_(X) and antisense N. Overhangsare not shown in the P diagram. In some embodiments a 3′ AS overhangcorresponds to region Z, the duplex region corresponds to region X, andthe 3′ S strand overhang corresponds to region Y, as described elsewhereherein. (The diagram is not meant to imply maximum or minimum lengths,on which guidance is provided elsewhere herein.)

It is preferred that pairings which decrease the propensity to form aduplex are used at 1 or more of the positions in the duplex at the 5′end of the AS strand. The terminal pair (the most 5′ pair in terms ofthe AS strand) is designated as P⁻¹, and the subsequent pairingpositions (going in the 3′ direction in terms of the AS strand) in theduplex are designated, P⁻², P⁻³, P⁻⁴, P⁻⁵, and so on. The preferredregion in which to modify to modulate duplex formation is at P⁻⁵ throughP⁻¹, more preferably P⁻⁴ through P⁻¹, more preferably P⁻³ through P⁻¹.Modification at P⁻1, is particularly preferred, alone or withmodification(s) other position(s), e.g., any of the positions justidentified. It is preferred that at least 1, and more preferably 2, 3,4, or 5 of the pairs of one of the recited regions be chosenindependently from the group of:

A:U

G:U

I:C

mismatched pairs, e.g., non-canonical or other than canonical pairingsor pairings which include a universal base.

In preferred embodiments the change in subunit needed to achieve apairing which promotes dissociation will be made in the sense strand,though in some embodiments the change will be made in the antisensestrand.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are pairs which promote disociation.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are A:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are G:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are I:C.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are mismatched pairs, e.g., non-canonical or other thancanonical pairings pairings.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are pairings which include a universal base.

Increasing the Stability of the AS 3′ End of the Duplex

Subunit pairs can be ranked on the basis of their propensity to promotestability and inhibit dissociation or melting (e.g., on the free energyof association or dissociation of a particular pairing, the simplestapproach is to examine the pairs on an individual pair basis, thoughnext neighbor or similar analysis can also be used). In terms ofpromoting duplex stability:

-   -   G:C is preferred over A:U    -   Watson-Crick matches (A:T, A:U, G:C) are preferred over        non-canonical or other than canonical pairings    -   analogs that increase stability are preferred over Watson-Crick        matches (A:T, A:U, G:C)    -   2-amino-A:U is preferred over A:U 2-thio U or 5 Me-thio-U:A are        preferred over U:A    -   G-clamp (an analog of C having 4 hydrogen bonds):G is preferred        over C:G    -   guanadinium-G-clamp:G is preferred over C:G    -   psuedo uridine:A is preferred over U:A

sugar modifications, e.g., 2′ modifications, e.g., 2′F, ENA, or LNA,which enhance binding are preferred over non-modified moieties and canbe present on one or both strands to enhance stability of the duplex. Itis preferred that pairings which increase the propensity to form aduplex are used at 1 or more of the positions in the duplex at the 3′end of the AS strand. The terminal pair (the most 3′ pair in terms ofthe AS strand) is designated as P₁, and the subsequent pairing positions(going in the 5′ direction in terms of the AS strand) in the duplex aredesignated, P₂, P₃, P₄, P₅, and so on. The preferred region in which tomodify to modulate duplex formation is at P₅ through P₁, more preferablyP₄ through P₁, more preferably P₃ through P₁. Modification at P₁, isparticularly preferred, alone or with mdification(s) at otherposition(s), e.g., any of the positions just identified. It is preferredthat at least 1, and more preferably 2, 3, 4, or 5 of the pairs of therecited regions be chosen independently from the group of:

G:C

a pair having an analog that increases stability over Watson-Crickmatches (A:T, A:U, G:C)

2-amino-A:U

2-thio U or 5 Me-thio-U:A

G-clamp (an analog of C having 4 hydrogen bonds):G

guanadinium-G-clamp:G

psuedo uridine:A

a pair in which one or both subunits has a sugar modification, e.g., a2′ modification, e.g., 2′F, ENA, or LNA, which enhance binding.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are pairs which promote duplex stability.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are G:C.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are a pair having an analog that increases stability overWatson-Crick matches.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are 2-amino-A:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are 2-thio U or 5 Me-thio-U:A.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are G-clamp:G.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are

guanidinium-G-clamp: G.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are psuedo uridine:A.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are a pair in which one or both subunits has a sugarmodification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, whichenhances binding.

G-clamps and guanidinium G-clamps are discussed in the followingreferences: Holmes and Gait, “The Synthesis of 2′-O-Methyl G-ClampContaining Oligonucleotides and Their Inhibition of the HIV-1 Tat-TARInteraction,” Nucleosides, Nucleotides & Nucleic Acids, 22:1259-1262,2003; Holmes et al., “Steric inhibition of human immunodeficiency virustype-1

Tat-dependent trans-activation in vitro and in cells by oligonucleotidescontaining 2′-O-methyl G-clamp ribonucleoside analogues,” Nucleic AcidsResearch, 31:2759-2768, 2003; Wilds, et al., “Structural basis forrecognition of guanosine by a synthetic tricyclic cytosine analogue:Guanidinium G-clamp,” Helvetica Chimica Acta, 86:966-978, 2003; Rajeev,et al., “High-Affinity Peptide Nucleic Acid Oligomers ContainingTricyclic Cytosine Analogues,” Organic Letters, 4:4395-4398, 2002;Ausin, et al., “Synthesis of Amino- and Guanidino-G-Clamp PNA Monomers,”Organic Letters, 4:4073-4075, 2002; Maier et al., “Nuclease resistanceof oligonucleotides containing the tricyclic cytosine analoguesphenoxazine and 9-(2-aminoethoxy)-phenoxazine (“G-clamp”) and origins oftheir nuclease resistance properties,” Biochemistry, 41:1323-7, 2002;Flanagan, et al., “A cytosine analog that confers enhanced potency toantisense oligonucleotides,” Proceedings Of The National Academy OfSciences Of The United States Of America, 96:3513-8, 1999.

Simultaneously Decreasing the Stability of the AS 5′ End of the Duplexand Increasing the Stability of the AS 3′ End of the Duplex

As is discussed above, an iRNA agent can be modified to both decreasethe stability of the AS 5′ end of the duplex and increase the stabilityof the AS 3′ end of the duplex. This can be effected by combining one ormore of the stability decreasing modifications in the AS 5′ end of theduplex with one or more of the stability increasing modifications in theAS 3′ end of the duplex. Accordingly a preferred embodiment includesmodification in P⁻⁵ through P⁻¹, more preferably P⁻⁴ through P⁻¹ andmore preferably P⁻³ through P⁻¹. Modification at P⁻¹, is particularlypreferred, alone or with other position, e.g., the positions justidentified. It is preferred that at least 1, and more preferably 2, 3,4, or 5 of the pairs of one of the recited regions of the AS 5′ end ofthe duplex region be chosen independently from the group of:

A:U

G:U

I:C

mismatched pairs, e.g., non-canonical or other than canonical pairingswhich include a universal base; and

a modification in P₅ through P₁, more preferably P₄ through P₁ and morepreferably P₃ through P₁. Modification at P₁, is particularly preferred,alone or with other position, e.g., the positions just identified. It ispreferred that at least 1, and more preferably 2, 3, 4, or 5 of thepairs of one of the recited regions of the AS 3′ end of the duplexregion be chosen independently from the group of:

G:C

a pair having an analog that increases stability over Watson-Crickmatches (A:T, A:U, G:C)

2-amino-A:U

2-thio U or 5 Me-thio-U:A

G-clamp (an analog of C having 4 hydrogen bonds):G

guanadinium-G-clamp:G

psuedo uridine:A

a pair in which one or both subunits has a sugar modification, e.g., a2′ modification, e.g., 2′F, ENA, or LNA, which enhance binding.

The invention also includes methods of selecting and making iRNA agentshaving DMTDS. E.g., when screening a target sequence for candidatesequences for use as iRNA agents one can select sequences having a DMTDSproperty described herein or one which can be modified, preferably withas few changes as possible, especially to the AS strand, to provide adesired level of DMTDS.

The invention also includes, providing a candidate iRNA agent sequence,and modifying at least one P in P⁻⁵ through P⁻¹ and/or at least one P inP₅ through P₁ to provide a DMTDS iRNA agent.

DMTDS iRNA agents can be used in any method described herein, e.g., tosilence any gene disclosed herein, to treat any disorder describedherein, in any formulation described herein, and generally in and/orwith the methods and compositions described elsewhere herein. DMTDS iRNAagents can incorporate other modifications described herein, e.g., theattachment of targeting agents or the inclusion of modifications whichenhance stability, e.g., the inclusion of nuclease resistant monomers orthe inclusion of single strand overhangs (e.g., 3′ AS overhangs and/or3′ S strand overhangs) which self associate to form intrastrand duplexstructure. Preferably these iRNA agents will have an architecturedescribed herein.

Other Embodiments

An RNA, e.g., an iRNA agent, can be produced in a cell in vivo, e.g.,from exogenous DNA templates that are delivered into the cell. Forexample, the DNA templates can be inserted into vectors and used as genetherapy vectors. Gene therapy vectors can be delivered to a subject by,for example, intravenous injection, local administration (U.S. Pat. No.5,328,470), or by stereotactic injection (see, e.g., Chen et al., Proc.Natl. Acad. Sci. USA 91:3054-3057, 1994). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. The DNA templates, for example, caninclude two transcription units, one that produces a transcript thatincludes the top strand of an iRNA agent and one that produces atranscript that includes the bottom strand of an iRNA agent. When thetemplates are transcribed, the iRNA agent is produced, and processedinto sRNA agent fragments that mediate gene silencing.

In vivo Delivery

An iRNA agent can be linked, e.g., noncovalently linked to a polymer forthe efficient delivery of the iRNA agent to a subject, e.g., a mammal,such as a human. The iRNA agent can, for example, be complexed withcyclodextrin. Cyclodextrins have been used as delivery vehicles oftherapeutic compounds. Cyclodextrins can form inclusion complexes withdrugs that are able to fit into the hydrophobic cavity of thecyclodextrin. In other examples, cyclodextrins form non-covalentassociations with other biologically active molecules such asoligonucleotides and derivatives thereof. The use of cyclodextrinscreates a water-soluble drug delivery complex, that can be modified withtargeting or other functional groups. Cyclodextrin cellular deliverysystem for oligonucleotides described in U.S. Pat. No. 5,691,316, whichis hereby incorporated by reference, are suitable for use in methods ofthe invention. In this system, an oligonucleotide is noncovalentlycomplexed with a cyclodextrin, or the oligonucleotide is covalentlybound to adamantine which in turn is non-covalently associated with acyclodextrin.

The delivery molecule can include a linear cyclodextrin copolymer or alinear oxidized cyclodextrin copolymer having at least one ligand boundto the cyclodextrin copolymer. Delivery systems, as described in U.S.Pat. No. 6,509,323, herein incorporated by reference, are suitable foruse in methods of the invention. An iRNA agent can be bound to thelinear cyclodextrin copolymer and/or a linear oxidized cyclodextrincopolymer. Either or both of the cyclodextrin or oxidized cyclodextrincopolymers can be crosslinked to another polymer and/or bound to aligand.

A composition for iRNA delivery can employ an “inclusion complex,” amolecular compound having the characteristic structure of an adduct. Inthis structure, the “host molecule” spatially encloses at least part ofanother compound in the delivery vehicle. The enclosed compound (the“guest molecule”) is situated in the cavity of the host molecule withoutaffecting the framework structure of the host. A “host” is preferablycyclodextrin, but can be any of the molecules suggested in U.S. PatentPubl. 2003/0008818, herein incorporated by reference.

Cyclodextrins can interact with a variety of ionic and molecularspecies, and the resulting inclusion compounds belong to the class of“host-guest” complexes. Within the host-guest relationship, the bindingsites of the host and guest molecules should be complementary in thestereoelectronic sense. A composition of the invention can contain atleast one polymer and at least one therapeutic agent, generally in theform of a particulate composite of the polymer and therapeutic agent,e.g., the iRNA agent. The iRNA agent can contain one or more complexingagents. At least one polymer of the particulate composite can interactwith the complexing agent in a host-guest or a guest-host interaction toform an inclusion complex between the polymer and the complexing agent.The polymer and, more particularly, the complexing agent can be used tointroduce functionality into the composition. For example, at least onepolymer of the particulate composite has host functionality and forms aninclusion complex with a complexing agent having guest functionality.Alternatively, at least one polymer of the particulate composite hasguest functionality and forms an inclusion complex with a complexingagent having host functionality. A polymer of the particulate compositecan also contain both host and guest functionalities and form inclusioncomplexes with guest complexing agents and host complexing agents. Apolymer with functionality can, for example, facilitate cell targetingand/or cell contact (e.g., targeting or contact to a kidney cell),intercellular trafficking, and/or cell entry and release.

Upon forming the particulate composite, the iRNA agent may or may notretain its biological or therapeutic activity. Upon release from thetherapeutic composition, specifically, from the polymer of theparticulate composite, the activity of the iRNA agent is restored.Accordingly, the particulate composite advantageously affords the iRNAagent protection against loss of activity due to, for example,degradation and offers enhanced bioavailability. Thus, a composition maybe used to provide stability, particularly storage or solutionstability, to an iRNA agent or any active chemical compound. The iRNAagent may be further modified with a ligand prior to or afterparticulate composite or therapeutic composition formation. The ligandcan provide further functionality. For example, the ligand can be atargeting moiety.

Physiological Effects

The iRNA agents described herein can be designed such that determiningtherapeutic toxicity is made easier by the complementarity of the iRNAagent with both a human and a non-human animal sequence. By thesemethods, an iRNA agent can consist of a sequence that is fullycomplementary to a nucleic acid sequence from a human and a nucleic acidsequence from at least one non-human animal, e.g., a non-human mammal,such as a rodent, ruminant or primate. For example, the non-human mammalcan be a mouse, rat, dog, pig, goat, sheep, cow, monkey, Pan paniscus,Pan troglodytes, Macaca mulatto, or Cynomolgus monkey. The sequence ofthe iRNA agent could be complementary to sequences within homologousgenes, e.g., oncogenes or tumor suppressor genes, of the non-humanmammal and the human. By determining the toxicity of the iRNA agent inthe non-human mammal, one can extrapolate the toxicity of the iRNA agentin a human. For a more strenuous toxicity test, the iRNA agent can becomplementary to a human and more than one, e.g., two or three or more,non-human animals.

The methods described herein can be used to correlate any physiologicaleffect of an iRNA agent on a human, e.g., any unwanted effect, such as atoxic effect, or any positive, or desired effect.

Delivery Module

The monomers and methods described herein can be used to prepare an RNA,e.g., an iRNA agent described herein, that can be used with a drugdelivery conjugate or module, such as those described herein and thosedescribed in copending, co-owned U.S. Provisional Application Ser. No.60/454,265, filed on Mar. 12, 2003, and International Application SerialNo. PCT/US04/07070, filed Mar. 8, 2004, both of which are herebyincorporated by reference.

The iRNA agents can be complexed to a delivery agent that features amodular complex. The complex can include a carrier agent linked to oneor more of (preferably two or more, more preferably all three of): (a) acondensing agent (e.g., an agent capable of attracting, e.g., binding, anucleic acid, e.g., through ionic or electrostatic interactions); (b) afusogenic agent (e.g., an agent capable of fusing and/or beingtransported through a cell membrane, e.g., an endosome membrane); and(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., alectin, glycoprotein, lipid or protein, e.g., an antibody, that binds toa specified cell type such as a kidney cell.

An iRNA agent, e.g., iRNA agent or sRNA agent described herein, can belinked, e.g., coupled or bound, to the modular complex. The iRNA agentcan interact with the condensing agent of the complex, and the complexcan be used to deliver an iRNA agent to a cell, e.g., in vitro or invivo. For example, the complex can be used to deliver an iRNA agent to asubject in need thereof, e.g., to deliver an iRNA agent to a subjecthaving a disorder, e.g., a disorder described herein, such as a diseaseor disorder of the kidney.

The fusogenic agent and the condensing agent can be different agents orthe one and the same agent. For example, a polyamino chain, e.g.,polyethyleneimine (PEI), can be the fusogenic and/or the condensingagent.

The delivery agent can be a modular complex. For example, the complexcan include a carrier agent linked to one or more of (preferably two ormore, more preferably all three of):

(a) a condensing agent (e.g., an agent capable of attracting, e.g.,binding, a nucleic acid, e.g., through ionic interaction),

(b) a fusogenic agent (e.g., an agent capable of fusing and/or beingtransported through a cell membrane, e.g., an endosome membrane), and

(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., alectin, glycoprotein, lipid or protein, e.g., an antibody, that binds toa specified cell type such as a kidney cell. A targeting group can be athyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A,Mucin carbohydrate, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,multivalent fucose, glycosylated polyaminoacids, multivalent galactose,transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid,cholesterol, a steroid, bile acid, folate, vitamin B12, biotin,Neproxin, or an RGD peptide or RGD peptide mimetic.

Carrier Agents

The carrier agent of a modular complex described herein can be asubstrate for attachment of one or more of: a condensing agent, afusogenic agent, and a targeting group. The carrier agent wouldpreferably lack an endogenous enzymatic activity. The agent wouldpreferably be a biological molecule, preferably a macromolecule.Polymeric biological carriers are preferred. It would also be preferredthat the carrier molecule be biodegradable.

The carrier agent can be a naturally occurring substance, such as aprotein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL),or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid); or lipid. The carrier moleculecan also be a recombinant or synthetic molecule, such as a syntheticpolymer, e.g., a synthetic polyamino acid. Examples of polyamino acidsinclude polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Other usefulcarrier molecules can be identified by routine methods.

A carrier agent can be characterized by one or more of: (a) is at least1 Da in size; (b) has at least 5 charged groups, preferably between 5and 5000 charged groups; (c) is present in the complex at a ratio of atleast 1:1 carrier agent to fusogenic agent; (d) is present in thecomplex at a ratio of at least 1:1 carrier agent to condensing agent;(e) is present in the complex at a ratio of at least 1:1 carrier agentto targeting agent.

Fusogenic Agents

A fusogenic agent of a modular complex described herein can be an agentthat is responsive to, e.g., changes charge depending on, the pHenvironment. Upon encountering the pH of an endosome, it can cause aphysical change, e.g., a change in osmotic properties which disrupts orincreases the permeability of the endosome membrane. Preferably, thefusogenic agent changes charge, e.g., becomes protonated, at pH lowerthan physiological range. For example, the fusogenic agent can becomeprotonated at pH 4.5-6.5. The fusogenic agent can serve to release theiRNA agent into the cytoplasm of a cell after the complex is taken up,e.g., via endocytosis, by the cell, thereby increasing the cellularconcentration of the iRNA agent in the cell.

In one embodiment, the fusogenic agent can have a moiety, e.g., an aminogroup, which, when exposed to a specified pH range, will undergo achange, e.g., in charge, e.g., protonation.

The change in charge of the fusogenic agent can trigger a change, e.g.,an osmotic change, in a vesicle, e.g., an endocytic vesicle, e.g., anendosome. For example, the fusogenic agent, upon being exposed to the pHenvironment of an endosome, will cause a solubility or osmotic changesubstantial enough to increase the porosity of (preferably, to rupture)the endosomal membrane.

The fusogenic agent can be a polymer, preferably a polyamino chain,e.g., polyethyleneimine (PEI). The PEI can be linear, branched,synthetic or natural. The PEI can be, e.g., alkyl substituted PEI, orlipid substituted PEI.

In other embodiments, the fusogenic agent can be polyhistidine,polyimidazole, polypyridine, polypropyleneimine, mellitin, or apolyacetal substance, e.g., a cationic polyacetal.

In some embodiment, the fusogenic agent can have an alpha helicalstructure. The fusogenic agent can be a membrane disruptive agent, e.g.,mellittin.

A fusogenic agent can have one or more of the following characteristics:(a) is at least 1 Da in size; (b) has at least 10 charged groups,preferably between 10 and 5000 charged groups, more preferably between50 and 1000 charged groups; (c) is present in the complex at a ratio ofat least 1:1fusogenic agent to carrier agent; (d) is present in thecomplex at a ratio of at least 1:1 fusogenic agent to condensing agent;(e) is present in the complex at a ratio of at least 1:1 fusogenic agentto targeting agent.

Other suitable fusogenic agents can be tested and identified by askilled artisan. The ability of a compound to respond to, e.g., changecharge depending on, the pH environment can be tested by routinemethods, e.g., in a cellular assay. For example, a test compound iscombined or contacted with a cell, and the cell is allowed to take upthe test compound, e.g., by endocytosis. An endosome preparation canthen be made from the contacted cells and the endosome preparationcompared to an endosome preparation from control cells. A change, e.g.,a decrease, in the endosome fraction from the contacted cell vs. thecontrol cell indicates that the test compound can function as afusogenic agent. Alternatively, the contacted cell and control cell canbe evaluated, e.g., by microscopy, e.g., by light or electronmicroscopy, to determine a difference in endosome population in thecells. The test compound can be labeled. In another type of assay, amodular complex described herein is constructed using one or more testor putative fusogenic agents. The modular complex can be constructedusing a labeled nucleic acid instead of the iRNA. The ability of thefusogenic agent to respond to, e.g., change charge depending on, the pHenvironment, once the modular complex is taken up by the cell, can beevaluated, e.g., by preparation of an endosome preparation, or bymicroscopy techniques, as described above. A two-step assay can also beperformed, wherein a first assay evaluates the ability of a testcompound alone to respond to, e.g., change charge depending on, the pHenvironment; and a second assay evaluates the ability of a modularcomplex that includes the test compound to respond to, e.g., changecharge depending on, the pH environment.

Condensing Agent

The condensing agent of a modular complex described herein can interactwith (e.g., attracts, holds, or binds to) an iRNA agent and act to (a)condense, e.g., reduce the size or charge of the iRNA agent and/or (b)protect the iRNA agent, e.g., protect the iRNA agent againstdegradation. The condensing agent can include a moiety, e.g., a chargedmoiety, that can interact with a nucleic acid, e.g., an iRNA agent,e.g., by ionic interactions. The condensing agent would preferably be acharged polymer, e.g., a polycationic chain. The condensing agent can bea polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quarternary salt of a polyamine, or an alpha helical peptide.

A condensing agent can have the following characteristics: (a) at least1 Da in size; (b) has at least 2 charged groups, preferably between 2and 100 charged groups; (c) is present in the complex at a ratio of atleast 1:1 condensing agent to carrier agent; (d) is present in thecomplex at a ratio of at least 1:1 condensing agent to fusogenic agent;(e) is present in the complex at a ratio of at least 1:1 condensingagent to targeting agent.

Other suitable condensing agents can be tested and identified by askilled artisan, e.g., by evaluating the ability of a test agent tointeract with a nucleic acid, e.g., an iRNA agent. The ability of a testagent to interact with a nucleic acid, e.g., an iRNA agent, e.g., tocondense or protect the iRNA agent, can be evaluated by routinetechniques. In one assay, a test agent is contacted with a nucleic acid,and the size and/or charge of the contacted nucleic acid is evaluated bya technique suitable to detect changes in molecular mass and/or charge.Such techniques include non-denaturing gel electrophoresis,immunological methods, e.g., immunoprecipitation, gel filtration, ionicinteraction chromatography, and the like. A test agent is identified asa condensing agent if it changes the mass and/or charge (preferablyboth) of the contacted nucleic acid, compared to a control. A two-stepassay can also be performed, wherein a first assay evaluates the abilityof a test compound alone to interact with, e.g., bind to, e.g., condensethe charge and/or mass of, a nucleic cid; and a second assay evaluatesthe ability of a modular complex that includes the test compound tointeract with, e.g., bind to, e.g., condense the charge and/or mass of,a nucleic acid.

Amphipathic Delivery Agents

The monomers and methods described herein can be used to prepare an RNA,e.g., an iRNA agent described herein, that can be used with anamphipathic delivery conjugate or module, such as those described hereinand those described in copending, co-owned U.S. Provisional ApplicationSer. No. 60/455,050, filed on Mar. 13, 2003, and InternationalApplication Serial No. PCT/US04/07070, filed Mar. 8, 2004, which ishereby incorporated by reference.

An amphipathic molecule is a molecule having a hydrophobic and ahydrophilic region. Such molecules can interact with (e.g., penetrate ordisrupt) lipids, e.g., a lipid bylayer of a cell. As such, they canserve as delivery agent for an associated (e.g., bound) iRNA (e.g., aniRNA or sRNA described herein). A preferred amphipathic molecule to beused in the compositions described herein (e.g., the amphipathic iRNAconstructs descriebd herein) is a polymer. The polymer may have asecondary structure, e.g., a repeating secondary structure.

One example of an amphipathic polymer is an amphipathic polypeptide,e.g., a polypeptide having a secondary structure such that thepolypeptide has a hydrophilic and a hybrophobic face. The design ofamphipathic peptide structures (e.g., alphα-helical polypeptides) isroutine to one of skill in the art. For example, the followingreferences provide guidance: Grell et al. (2001) “Protein design andfolding: template trapping of self-assembled helical bundles” J Pept Sci7(3):146-51; Chen et al. (2002) “Determination of stereochemistrystability coefficients of amino acid side-chains in an amphipathicalpha-helix” J Pept Res 59(1):18-33; Iwata et al. (1994) “Design andsynthesis of amphipathic 3(10)-helical peptides and their interactionswith phospholipid bilayers and ion channel formation” J Biol Chem269(7):4928-33; Cornut et al. (1994) “The amphipathic alpha-helixconcept. Application to the de novo design of ideally amphipathic Leu,Lys peptides with hemolytic activity higher than that of melittin” FEBSLett 349(1):29-33; Negrete et al. (1998) “Deciphering the structuralcode for proteins: helical propensities in domain classes andstatistical multiresidue information in alpha-helices,” Protein Sci7(6):1368-79.

Another example of an amphipathic polymer is a polymer made up of two ormore amphipathic subunits, e.g., two or more subunits containing cyclicmoieties (e.g., a cyclic moiety having one or more hydrophilic groupsand one or more hydrophobic groups). For example, the subunit maycontain a steroid, e.g., cholic acid; or a aromatic moiety. Suchmoieties preferably can exhibit atropisomerism, such that they can formopposing hydrophobic and hydrophilic faces when in a polymer structure.

The ability of a putative amphipathic molecule to interact with a lipidmembrane, e.g., a cell membrane, can be tested by routine methods, e.g.,in a cell free or cellular assay. For example, a test compound iscombined or contacted with a synthetic lipid bilayer, a cellularmembrane fraction, or a cell, and the test compound is evaluated for itsability to interact with, penetrate or disrupt the lipid bilayer, cellmembrane or cell. The test compound can labeled in order to detect theinteraction with the lipid bilayer, cell membrane or cell. In anothertype of assay, the test compound is linked to a reporter molecule or aniRNA agent (e.g., an iRNA or sRNA described herein) and the ability ofthe reporter molecule or iRNA agent to penetrate the lipid bilayer, cellmembrane or cell is evaluated. A two-step assay can also be performed,wherein a first assay evaluates the ability of a test compound alone tointeract with a lipid bilayer, cell membrane or cell; and a second assayevaluates the ability of a construct (e.g., a construct describedherein) that includes the test compound and a reporter or iRNA agent tointeract with a lipid bilayer, cell membrane or cell. An amphipathicpolymer useful in the compositions described herein has at least 2,preferably at least 5, more preferably at least 10, 25, 50, 100, 200,500, 1000, 2000, 50000 or more subunits (e.g., amino acids or cyclicsubunits). A single amphipathic polymer can be linked to one or more,e.g., 2, 3, 5, 10 or more iRNA agents (e.g., iRNA or sRNA agentsdescribed herein). In some embodiments, an amphipathic polymer cancontain both amino acid and cyclic subunits, e.g., aromatic subunits.

The invention features a composition that includes an iRNA agent (e.g.,an iRNA or sRNA described herein) in association with an amphipathicmolecule. Such compositions may be referred to herein as “amphipathiciRNA constructs.” Such compositions and constructs are useful in thedelivery or targeting of iRNA agents, e.g., delivery or targeting ofiRNA agents to a cell. While not wanting to be bound by theory, suchcompositions and constructs can increase the porosity of, e.g., canpenetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g.,to allow entry of the iRNA agent into a cell.

In one aspect, the invention relates to a composition comprising an iRNAagent (e.g., an iRNA or sRNA agent described herein) linked to anamphipathic molecule. The iRNA agent and the amphipathic molecule may beheld in continuous contact with one another by either covalent ornoncovalent linkages.

The amphipathic molecule of the composition or construct is preferablyother than a phospholipid, e.g., other than a micelle, membrane ormembrane fragment.

The amphipathic molecule of the composition or construct is preferably apolymer. The polymer may include two or more amphipathic subunits. Oneor more hydrophilic groups and one or more hydrophobic groups may bepresent on the polymer. The polymer may have a repeating secondarystructure as well as a first face and a second face. The distribution ofthe hydrophilic groups and the hydrophobic groups along the repeatingsecondary structure can be such that one face of the polymer is ahydrophilic face and the other face of the polymer is a hydrophobicface.

The amphipathic molecule can be a polypeptide, e.g., a polypeptidecomprising an α-helical conformation as its secondary structure.

In one embodiment, the amphipathic polymer includes one or more subunitscontaining one or more cyclic moiety (e.g., a cyclic moiety having oneor more hydrophilic groups and/or one or more hydrophobic groups). Inone embodiment, the polymer is a polymer of cyclic moieties such thatthe moieties have alternating hydrophobic and hydrophilic groups. Forexample, the subunit may contain a steroid, e.g., cholic acid. Inanother example, the subunit may contain an aromatic moiety. Thearomatic moiety may be one that can exhibit atropisomerism, e.g., a2,2′-bis(substituted)-1-1′-binaphthyl or a 2,2′-bis(substituted)biphenyl. A subunit may include an aromatic moiety of Formula (M):

The invention features a composition that includes an iRNA agent (e.g.,an iRNA or sRNA described herein) in association with an amphipathicmolecule. Such compositions may be referred to herein as “amphipathiciRNA constructs.” Such compositions and constructs are useful in thedelivery or targeting of iRNA agents, e.g., delivery or targeting ofiRNA agents to a cell. While not wanting to be bound by theory, suchcompositions and constructs can increase the porosity of, e.g., canpenetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g.,to allow entry of the iRNA agent into a cell.

In one aspect, the invention relates to a composition comprising an iRNAagent (e.g., an iRNA or sRNA agent described herein) linked to anamphipathic molecule. The iRNA agent and the amphipathic molecule may beheld in continuous contact with one another by either covalent ornoncovalent linkages.

The amphipathic molecule of the composition or construct is preferablyother than a phospholipid, e.g., other than a micelle, membrane ormembrane fragment.

The amphipathic molecule of the composition or construct is preferably apolymer. The polymer may include two or more amphipathic subunits. Oneor more hydrophilic groups and one or more hydrophobic groups may bepresent on the polymer. The polymer may have a repeating secondarystructure as well as a first face and a second face. The distribution ofthe hydrophilic groups and the hydrophobic groups along the repeatingsecondary structure can be such that one face of the polymer is ahydrophilic face and the other face of the polymer is a hydrophobicface.

The amphipathic molecule can be a polypeptide, e.g., a polypeptidecomprising an α-helical conformation as its secondary structure.

In one embodiment, the amphipathic polymer includes one or more subunitscontaining one or more cyclic moiety (e.g., a cyclic moiety having oneor more hydrophilic groups and/or one or more hydrophobic groups). Inone embodiment, the polymer is a polymer of cyclic moieties such thatthe moieties have alternating hydrophobic and hydrophilic groups. Forexample, the subunit may contain a steroid, e.g., cholic acid. Inanother example, the subunit may contain an aromatic moiety. Thearomatic moiety may be one that can exhibit atropisomerism, e.g., a2,2′-bis(substituted)-1-1′-binaphthyl or a 2,2′-bis(substituted)biphenyl. A subunit may include an aromatic moiety of Formula (M):

Referring to Formula M, R₁ is C₁-C₁₀₀ alkyl optionally substituted witharyl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted withO, S, alkenyl or alkynyl; C₁-C₁₀₀ perfluoroalkyl; or OR₅.

R₂ is hydroxy; nitro; sulfate; phosphate; phosphate ester; sulfonicacid; OR₆; or C₁-C₁₀₀ alkyl optionally substituted with hydroxy, halo,nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonicacid, phosphate, phosphate ester, substituted or unsubstituted aryl,carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/oroptionally inserted with O, NH, S, S(O), SO₂, alkenyl, or alkynyl.

R₃ is hydrogen, or when taken together with R₄ forms a fused phenylring.

R₄ is hydrogen, or when taken together with R₃ forms a fused phenylring.

R₅ is C₁-C₁₀₀ alkyl optionally substituted with aryl, alkenyl, alkynyl,alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl;or C₁-C₁₀₀ perfluoroalkyl; and R₆ is C₁-C₁₀₀ alkyl optionallysubstituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl oralkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester,substituted or unsubstituted aryl, carboxyl, carboxylate, aminocarbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S,S(O), SO₂, alkenyl, or alkynyl.

Increasing Cellular Uptake of dsRNAs

A method of the invention that can include the administration of an iRNAagent and a drug that affects the uptake of the iRNA agent into thecell. The drug can be administered before, after, or at the same timethat the iRNA agent is administered. The drug can be covalently linkedto the iRNA agent. The drug can be, for example, a lipopolysaccharide,an activator of p38 MAP kinase, or an activator of NF-κB. The drug canhave a transient effect on the cell.

The drug can increase the uptake of the iRNA agent into the cell, forexample, by disrupting the cell's cytoskeleton, e.g., by disrupting thecell's microtubules, microfilaments, and/or intermediate filaments. Thedrug can be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin.

The drug can also increase the uptake of the iRNA agent into the cell byactivating an inflammatory response, for example. Exemplary drug's thatwould have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

iRNA Conjugates

An iRNA agent can be coupled, e.g., covalently coupled, to a secondagent. For example, an iRNA agent used to treat a particular disordercan be coupled to a second therapeutic agent, e.g., an agent other thanthe iRNA agent. The second therapeutic agent can be one which isdirected to the treatment of the same disorder. For example, in the caseof an iRNA used to treat a disorder characterized by unwanted cellproliferation, e.g., cancer, the iRNA agent can be coupled to a secondagent which has an anti-cancer effect. For example, it can be coupled toan agent which stimulates the immune system, e.g., a CpG motif, or moregenerally an agent that activates a toll-like receptor and/or increasesthe production of gamma interferon.

iRNa Production

An iRNA can be produced, e.g., in bulk, by a variety of methods.Exemplary methods include: organic synthesis and RNA cleavage, e.g., invitro cleavage.

Organic Synthesis

An iRNA can be made by separately synthesizing each respective strand ofa double-stranded RNA molecule. The component strands can then beannealed.

A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB(Uppsala Sweden), can be used to produce a large amount of a particularRNA strand for a given iRNA. The OligoPilotII reactor can efficientlycouple a nucleotide using only a 1.5 molar excess of a phosphoramiditenucleotide. To make an RNA strand, ribonucleotides amidites are used.Standard cycles of monomer addition can be used to synthesize the 21 to23 nucleotide strand for the iRNA. Typically, the two complementarystrands are produced separately and then annealed, e.g., after releasefrom the solid support and deprotection.

Organic synthesis can be used to produce a discrete iRNA species. Thecomplementary of the species to a particular target gene can beprecisely specified. For example, the species may be complementary to aregion that includes a polymorphism, e.g., a single nucleotidepolymorphism. Further the location of the polymorphism can be preciselydefined. In some embodiments, the polymorphism is located in an internalregion, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of thetermini.

dsRNA Cleavage

iRNAs can also be made by cleaving a larger ds iRNA. The cleavage can bemediated in vitro or in vivo. For example, to produce iRNAs by cleavagein vitro, the following method can be used:

In vitro transcription. dsRNA is produced by transcribing a nucleic acid(DNA) segment in both directions. For example, the HiScribe™ RNAitranscription kit (New England Biolabs) provides a vector and a methodfor producing a dsRNA for a nucleic acid segment that is cloned into thevector at a position flanked on either side by a T7 promoter. Separatetemplates are generated for T7 transcription of the two complementarystrands for the dsRNA. The templates are transcribed in vitro byaddition of T7 RNA polymerase and dsRNA is produced. Similar methodsusing PCR and/or other RNA polymerases (e.g., T3 or SP6 polymerase) canalso be used. In one embodiment, RNA generated by this method iscarefully purified to remove endotoxins that may contaminatepreparations of the recombinant enzymes.

In vitro cleavage. dsRNA is cleaved in vitro into iRNAs, for example,using a Dicer or comparable RNAse III-based activity. For example, thedsRNA can be incubated in an in vitro extract from Drosophila or usingpurified components, e.g. a purified RNAse or RISC complex (RNA-inducedsilencing complex). See, e.g., Ketting et al. Genes Dev 2001 Oct. 15;15(20):2654-9. and Hammond Science 2001 Aug. 10; 293(5532):1146-50.

dsRNA cleavage generally produces a plurality of iRNA species, eachbeing a particular 21 to 23 nt fragment of a source dsRNA molecule. Forexample, iRNAs that include sequences complementary to overlappingregions and adjacent regions of a source dsRNA molecule may be present.

Regardless of the method of synthesis, the iRNA preparation can beprepared in a solution (e.g., an aqueous and/or organic solution) thatis appropriate for formulation. For example, the iRNA preparation can beprecipitated and redissolved in pure double-distilled water, andlyophilized. The dried iRNA can then be resuspended in a solutionappropriate for the intended formulation process.

Synthesis of modified and nucleotide surrogate iRNA agents is discussedbelow.

Formulation

The iRNA agents described herein can be formulated for administration toa subject

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention.

A formulated iRNA composition can assume a variety of states. In someexamples, the composition is at least partially crystalline, uniformlycrystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10%water). In another example, the iRNA is in an aqueous phase, e.g., in asolution that includes water.

The aqueous phase or the crystalline compositions can, e.g., beincorporated into a delivery vehicle, e.g., a liposome (particularly forthe aqueous phase) or a particle (e.g., a microparticle as can beappropriate for a crystalline composition). Generally, the iRNAcomposition is formulated in a manner that is compatible with theintended method of administration (see, below).

In particular embodiments, the composition is prepared by at least oneof the following methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

A iRNA preparation can be formulated in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes a iRNA,e.g., a protein that complexes with iRNA to form an iRNP. Still otheragents include chelators, e.g., EDTA (e.g., to remove divalent cationssuch as Mg²+), salts, RNAse inhibitors (e.g., a broad specificity RNAseinhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA preparation includes another iRNA agent,e.g., a second iRNA that can mediated RNAi with respect to a secondgene, or with respect to the same gene. Still other preparation caninclude at least 3, 5, ten, twenty, fifty, or a hundred or moredifferent iRNA species. Such iRNAs can mediated RNAi with respect to asimilar number of different genes.

In one embodiment, the iRNA preparation includes at least a secondtherapeutic agent (e.g., an agent other than an RNA or a DNA). Forexample, a iRNA composition for the treatment of a viral disease, e.g.HIV, might include a known antiviral agent (e.g., a protease inhibitoror reverse transcriptase inhibitor). In another example, a iRNAcomposition for the treatment of a cancer might further comprise achemotherapeutic agent. Exemplary formulations are discussed below:

Liposomes

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA s agents, and such practice is within the invention. AniRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof) preparation can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA composition. The lipophilic material isolates theaqueous interior from an aqueous exterior, which typically does notinclude the iRNA composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the iRNA are delivered into the cell where theiRNA can specifically bind to a target RNA and can mediate RNAi. In somecases the liposomes are also specifically targeted, e.g., to direct theiRNA to particular cell types, e.g., to cells of the kidney, such asthose described herein.

A liposome containing a iRNA can be prepared by a variety of methods.

In one example, the lipid component of a liposome is dissolved in adetergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNApreparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the iRNA andcondense around the iRNA to form a liposome. After condensation, thedetergent is removed, e.g., by dialysis, to yield a liposomalpreparation of iRNA.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Further description of methods for producing stable polynucleotidedelivery vehicles, which incorporate a polynucleotide/cationic lipidcomplex as structural components of the delivery vehicle, are describedin, e.g., WO 96/37194. Liposome formation can also include one or moreaspects of exemplary methods described in Feigner, P. L. et al., Proc.Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S.Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson,et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl.Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; andFukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques forpreparing lipid aggregates of appropriate size for use as deliveryvehicles include sonication and freeze-thaw plus extrusion (see, e.g.,Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidizationcan be used when consistently small (50 to 200 nm) and relativelyuniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta775:169, 1984). These methods are readily adapted to packaging iRNApreparations into liposomes.

Liposomes that are pH-sensitive or negatively-charged, entrap nucleicacid molecules rather than complex with them. Since both the nucleicacid molecules and the lipid are similarly charged, repulsion ratherthan complex formation occurs. Nevertheless, some nucleic acid moleculesare entrapped within the aqueous interior of these liposomes.pH-sensitive liposomes have been used to deliver DNA encoding thethymidine kinase gene to cell monolayers in culture. Expression of theexogenous gene was detected in the target cells (Zhou et al., Journal ofControlled Release, 19, (1992) 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550,1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human GeneTher. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J.11:417, 1992.

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver iRNAs to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated iRNAs in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of iRNA (see, e.g., Felgner,P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S.Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer iRNA, into the skin. In some implementations,liposomes are used for delivering iRNA to epidermal cells and also toenhance the penetration of iRNA into dermal tissues, e.g., into skin.For example, the liposomes can be applied topically. Topical delivery ofdrugs formulated as liposomes to the skin has been documented (see,e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino,R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T.et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176,1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527,1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith iRNA are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include iRNA can bedelivered, for example, subcutaneously by infection in order to deliveriRNA to keratinocytes in the skin. In order to cross intact mammalianskin, lipid vesicles must pass through a series of fine pores, each witha diameter less than 50 nm, under the influence of a suitabletransdermal gradient. In addition, due to the lipid properties, thesetransferosomes can be self-optimizing (adaptive to the shape of pores,e.g., in the skin), self-repairing, and can frequently reach theirtargets without fragmenting, and often self-loading. The iRNA agents caninclude an RRMS tethered to a moiety which improves association with aliposome.

Surfactants

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention.Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes (see above). iRNA (or aprecursor, e.g., a larger dsRNA which can be processed into a iRNA, or aDNA which encodes a iRNA or precursor) compositions can include asurfactant. In one embodiment, the iRNA is formulated as an emulsionthat includes a surfactant. The most common way of classifying andranking the properties of the many different types of surfactants, bothnatural and synthetic, is by the use of the hydrophile/lipophile balance(HLB). The nature of the hydrophilic group provides the most usefulmeans for categorizing the different surfactants used in formulations(Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., NewYork, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant.

Nonionic surfactants find wide application in pharmaceutical productsand are usable over a wide range of pH values. In general their HLBvalues range from 2 to about 18 depending on their structure. Nonionicsurfactants include nonionic esters such as ethylene glycol esters,propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitanesters, sucrose esters, and ethoxylated esters. Nonionic alkanolamidesand ethers such as fatty alcohol ethoxylates, propoxylated alcohols, andethoxylated/propoxylated block polymers are also included in this class.The polyoxyethylene surfactants are the most popular members of thenonionic surfactant class.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Micelles and other Membranous Formulations

For ease of exposition the micelles and other formulations, compositionsand methods in this section are discussed largely with regard tounmodified iRNA agents. It should be understood, however, that thesemicelles and other formulations, compositions and methods can bepracticed with other iRNA agents, e.g., modified iRNA agents, and suchpractice is within the invention. The iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof)) composition can be provided as a micellarformulation. “Micelles” are defined herein as a particular type ofmolecular assembly in which amphipathic molecules are arranged in aspherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the iRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing is preferred in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe iRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the iRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e. there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g. through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

The preferred propellants are hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether.Even more preferred is HFA 134a (1,1,1,2 tetrafluoroethane).

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g. atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

The iRNA agents can include an RRMS tethered to a moiety which improvesassociation with a micelle or other membranous formulation.

Particles

For ease of exposition the particles, formulations, compositions andmethods in this section are discussed largely with regard to unmodifiediRNA agents. It should be understood, however, that these particles,formulations, compositions and methods can be practiced with other iRNAagents, e.g., modified iRNA agents, and such practice is within theinvention. In another embodiment, an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into a sRNA agent, or a DNA which encodes an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursorthereof) preparations may be incorporated into a particle, e.g., amicroparticle. Microparticles can be produced by spray-drying, but mayalso be produced by other methods including lyophilization, evaporation,fluid bed drying, vacuum drying, or a combination of these techniques.See below for further description.

Sustained-Release Formulations.

An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g.,a precursor, e.g., a larger iRNA agent which can be processed into asRNA agent, or a DNA which encodes an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, or precursor thereof)described herein can be formulated for controlled, e.g., slow release.Controlled release can be achieved by disposing the iRNA within astructure or substance which impedes its release. E.g., iRNA can bedisposed within a porous matrix or in an erodable matrix, either ofwhich allow release of the iRNA over a period of time.

Polymeric particles, e.g., polymeric in microparticles can be used as asustained-release reservoir of iRNA that is taken up by cells onlyreleased from the microparticle through biodegradation. The polymericparticles in this embodiment should therefore be large enough topreclude phagocytosis (e.g., larger than 10 μm and preferably largerthan 20 μm). Such particles can be produced by the same methods to makesmaller particles, but with less vigorous mixing of the first and secondemulsions. That is to say, a lower homogenization speed, vortex mixingspeed, or sonication setting can be used to obtain particles having adiameter around 100 μm rather than 10 μm. The time of mixing also can bealtered.

Larger microparticles can be formulated as a suspension, a powder, or animplantable solid, to be delivered by intramuscular, subcutaneous,intradermal, intravenous, or intraperitoneal injection; via inhalation(intranasal or intrapulmonary); orally; or by implantation. Theseparticles are useful for delivery of any iRNA when slow release over arelatively long term is desired. The rate of degradation, andconsequently of release, varies with the polymeric formulation.

Microparticles preferably include pores, voids, hollows, defects orother interstitial spaces that allow the fluid suspension medium tofreely permeate or perfuse the particulate boundary. For example, theperforated microstructures can be used to form hollow, porous spraydried microspheres.

Polymeric particles containing iRNA (e.g., a sRNA) can be made using adouble emulsion technique, for instance. First, the polymer is dissolvedin an organic solvent. A preferred polymer is polylactic-co-glycolicacid (PLGA), with a lactic/glycolic acid weight ratio of 65:35, 50:50,or 75:25. Next, a sample of nucleic acid suspended in aqueous solutionis added to the polymer solution and the two solutions are mixed to forma first emulsion. The solutions can be mixed by vortexing or shaking,and in a preferred method, the mixture can be sonicated. Most preferableis any method by which the nucleic acid receives the least amount ofdamage in the form of nicking, shearing, or degradation, while stillallowing the formation of an appropriate emulsion. For example,acceptable results can be obtained with a Vibra-cell model VC-250sonicator with a ⅛″ microtip probe, at setting #3.

Spray-Drying.

An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g.,a precursor, e.g., a larger iRNA agent which can be processed into asRNA agent, or a DNA which encodes an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, or precursor thereof)) can beprepared by spray drying. Spray dried iRNA can be administered to asubject or be subjected to further formulation. A pharmaceuticalcomposition of iRNA can be prepared by spray drying a homogeneousaqueous mixture that includes a iRNA under conditions sufficient toprovide a dispersible powdered composition, e.g., a pharmaceuticalcomposition. The material for spray drying can also include one or moreof: a pharmaceutically acceptable excipient, or adispersibility-enhancing amount of a physiologically acceptable,water-soluble protein. The spray-dried product can be a dispersiblepowder that includes the iRNA.

Spray drying is a process that converts a liquid or slurry material to adried particulate form. Spray drying can be used to provide powderedmaterial for various administrative routes including inhalation. See,for example, M. Sacchetti and M. M. Van Oort in: Inhalation Aerosols:Physical and Biological Basis for Therapy, A. J. Hickey, ed. MarcelDekkar, New York, 1996.

Spray drying can include atomizing a solution, emulsion, or suspensionto form a fine mist of droplets and drying the droplets. The mist can beprojected into a drying chamber (e.g., a vessel, tank, tubing, or coil)where it contacts a drying gas. The mist can include solid or liquidpore forming agents. The solvent and pore forming agents evaporate fromthe droplets into the drying gas to solidify the droplets,simultaneously forming pores throughout the solid. The solid (typicallyin a powder, particulate form) then is separated from the drying gas andcollected.

Spray drying includes bringing together a highly dispersed liquid, and asufficient volume of air (e.g., hot air) to produce evaporation anddrying of the liquid droplets. The preparation to be spray dried can beany solution, course suspension, slurry, colloidal dispersion, or pastethat may be atomized using the selected spray drying apparatus.Typically, the feed is sprayed into a current of warm filtered air thatevaporates the solvent and conveys the dried product to a collector. Thespent air is then exhausted with the solvent. Several different types ofapparatus may be used to provide the desired product. For example,commercial spray dryers manufactured by Buchi Ltd. or Niro Corp. caneffectively produce particles of desired size.

Spray-dried powdered particles can be approximately spherical in shape,nearly uniform in size and frequently hollow. There may be some degreeof irregularity in shape depending upon the incorporated medicament andthe spray drying conditions. In many instances the dispersion stabilityof spray-dried microspheres appears to be more effective if an inflatingagent (or blowing agent) is used in their production. Particularlypreferred embodiments may comprise an emulsion with an inflating agentas the disperse or continuous phase (the other phase being aqueous innature). An inflating agent is preferably dispersed with a surfactantsolution, using, for instance, a commercially available microfluidizerat a pressure of about 5000 to 15,000 psi. This process forms anemulsion, preferably stabilized by an incorporated surfactant, typicallycomprising submicron droplets of water immiscible blowing agentdispersed in an aqueous continuous phase. The formation of suchdispersions using this and other techniques are common and well known tothose in the art. The blowing agent is preferably a fluorinated compound(e.g. perfluorohexane, perfluorooctyl bromide, perfluorodecalin,perfluorobutyl ethane) which vaporizes during the spray-drying process,leaving behind generally hollow, porous aerodynamically lightmicrospheres. As will be discussed in more detail below, other suitableblowing agents include chloroform, freons, and hydrocarbons. Nitrogengas and carbon dioxide are also contemplated as a suitable blowingagent.

Although the perforated microstructures are preferably formed using ablowing agent as described above, it will be appreciated that, in someinstances, no blowing agent is required and an aqueous dispersion of themedicament and surfactant(s) are spray dried directly. In such cases,the formulation may be amenable to process conditions (e.g., elevatedtemperatures) that generally lead to the formation of hollow, relativelyporous microparticles. Moreover, the medicament may possess specialphysicochemical properties (e.g., high crystallinity, elevated meltingtemperature, surface activity, etc.) that make it particularly suitablefor use in such techniques.

The perforated microstructures may optionally be associated with, orcomprise, one or more surfactants. Moreover, miscible surfactants mayoptionally be combined with the suspension medium liquid phase. It willbe appreciated by those skilled in the art that the use of surfactantsmay further increase dispersion stability, simplify formulationprocedures or increase bioavailability upon administration. Of coursecombinations of surfactants, including the use of one or more in theliquid phase and one or more associated with the perforatedmicrostructures are contemplated as being within the scope of theinvention. By “associated with or comprise” it is meant that thestructural matrix or perforated microstructure may incorporate, adsorb,absorb, be coated with or be formed by the surfactant.

Surfactants suitable for use include any compound or composition thataids in the formation and maintenance of the stabilized respiratorydispersions by forming a layer at the interface between the structuralmatrix and the suspension medium. The surfactant may comprise a singlecompound or any combination of compounds, such as in the case ofco-surfactants. Particularly preferred surfactants are substantiallyinsoluble in the propellant, nonfluorinated, and selected from the groupconsisting of saturated and unsaturated lipids, nonionic detergents,nonionic block copolymers, ionic surfactants, and combinations of suchagents. It should be emphasized that, in addition to the aforementionedsurfactants, suitable (i.e. biocompatible) fluorinated surfactants arecompatible with the teachings herein and may be used to provide thedesired stabilized preparations.

Lipids, including phospholipids, from both natural and synthetic sourcesmay be used in varying concentrations to form a structural matrix.Generally, compatible lipids comprise those that have a gel to liquidcrystal phase transition greater than about 40° C. Preferably, theincorporated lipids are relatively long chain (i.e. C₆-C₂₂) saturatedlipids and more preferably comprise phospholipids. Exemplaryphospholipids useful in the disclosed stabilized preparations compriseegg phosphatidylcholine, dilauroylphosphatidylcholine,dioleylphosphatidylcholine, dipalmitoylphosphatidyl-choline,disteroylphosphatidylcholine, short-chain phosphatidylcholines,phosphatidylethanolamine, dioleylphosphatidylethanolamine,phosphatidylserine, phosphatidylglycerol, phosphatidylinositol,glycolipids, ganglioside GM1, sphingomyelin, phosphatidic acid,cardiolipin; lipids bearing polymer chains such as, polyethylene glycol,chitin, hyaluronic acid, or polyvinylpyrrolidone; lipids bearingsulfonated mono-, di-, and polysaccharides; fatty acids such as palmiticacid, stearic acid, and oleic acid; cholesterol, cholesterol esters, andcholesterol hemisuccinate. Due to their excellent biocompatibilitycharacteristics, phospholipids and combinations of phospholipids andpoloxamers are particularly suitable for use in the stabilizeddispersions disclosed herein.

Compatible nonionic detergents comprise: sorbitan esters includingsorbitan trioleate (Spans™ 85), sorbitan sesquioleate, sorbitanmonooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitanmonolaurate, and polyoxyethylene (20) sorbitan monooleate, oleylpolyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, laurylpolyoxyethylene (4) ether, glycerol esters, and sucrose esters. Othersuitable nonionic detergents can be easily identified using McCutcheon'sEmulsifiers and Detergents (McPublishing Co., Glen Rock, N.J.).Preferred block copolymers include diblock and triblock copolymers ofpolyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic®F68), poloxamer 407 (Pluronic® F-127), and poloxamer 338. Ionicsurfactants such as sodium sulfosuccinate, and fatty acid soaps may alsobe utilized. In preferred embodiments, the microstructures may compriseoleic acid or its alkali salt.

In addition to the aforementioned surfactants, cationic surfactants orlipids are preferred especially in the case of delivery of an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof). Examples of suitablecationic lipids include: DOTMA,N—[-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium-chloride;DOTAP,1,2-dioleyloxy-3-(trimethylammonio)propane; and DOTB,1,2-dioleyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol. Polycationicamino acids such as polylysine, and polyarginine are also contemplated.

For the spraying process, such spraying methods as rotary atomization,pressure atomization and two-fluid atomization can be used. Examples ofthe devices used in these processes include “Parubisu [phoneticrendering] Mini-Spray GA-32” and “Parubisu Spray Drier DL-41”,manufactured by Yamato Chemical Co., or “Spray Drier CL-8,” “Spray DrierL-8,” “Spray Drier FL-12,” “Spray Drier FL-16” or “Spray Drier FL-20,”manufactured by Okawara Kakoki Co., can be used for the method ofspraying using rotary-disk atomizer.

While no particular restrictions are placed on the gas used to dry thesprayed material, it is recommended to use air, nitrogen gas or an inertgas. The temperature of the inlet of the gas used to dry the sprayedmaterials such that it does not cause heat deactivation of the sprayedmaterial. The range of temperatures may vary between about 50° C. toabout 200° C., preferably between about 50° C. and 100° C. Thetemperature of the outlet gas used to dry the sprayed material, may varybetween about 0° C. and about 150° C., preferably between 0° C. and 90°C., and even more preferably between 0° C. and 60° C.

The spray drying is done under conditions that result in substantiallyamorphous powder of homogeneous constitution having a particle size thatis respirable, a low moisture content and flow characteristics thatallow for ready aerosolization. Preferably the particle size of theresulting powder is such that more than about 98% of the mass is inparticles having a diameter of about 10 μm or less with about 90% of themass being in particles having a diameter less than 5 μm. Alternatively,about 95% of the mass will have particles with a diameter of less than10 μm with about 80% of the mass of the particles having a diameter ofless than 5 μm.

The dispersible pharmaceutical-based dry powders that include the iRNApreparation may optionally be combined with pharmaceutical carriers orexcipients which are suitable for respiratory and pulmonaryadministration. Such carriers may serve simply as bulking agents when itis desired to reduce the iRNA concentration in the powder which is beingdelivered to a patient, but may also serve to enhance the stability ofthe iRNA compositions and to improve the dispersibility of the powderwithin a powder dispersion device in order to provide more efficient andreproducible delivery of the iRNA and to improve handlingcharacteristics of the iRNA such as flowability and consistency tofacilitate manufacturing and powder filling.

Such carrier materials may be combined with the drug prior to spraydrying, i.e., by adding the carrier material to the purified bulksolution. In that way, the carrier particles will be formedsimultaneously with the drug particles to produce a homogeneous powder.Alternatively, the carriers may be separately prepared in a dry powderform and combined with the dry powder drug by blending. The powdercarriers will usually be crystalline (to avoid water absorption), butmight in some cases be amorphous or mixtures of crystalline andamorphous. The size of the carrier particles may be selected to improvethe flowability of the drug powder, typically being in the range from 25μm to 100 μm. A preferred carrier material is crystalline lactose havinga size in the above-stated range.

Powders prepared by any of the above methods will be collected from thespray dryer in a conventional manner for subsequent use. For use aspharmaceuticals and other purposes, it will frequently be desirable todisrupt any agglomerates which may have formed by screening or otherconventional techniques. For pharmaceutical uses, the dry powderformulations will usually be measured into a single dose, and the singledose sealed into a package. Such packages are particularly useful fordispersion in dry powder inhalers, as described in detail below.Alternatively, the powders may be packaged in multiple-dose containers.

Methods for spray drying hydrophobic and other drugs and components aredescribed in U.S. Pat. Nos. 5,000,888; 5,026,550; 4,670,419, 4,540,602;and 4,486,435. Bloch and Speison (1983) Pharm. Acta Hely 58:14-22teaches spray drying of hydrochlorothiazide and chlorthalidone(lipophilic drugs) and a hydrophilic adjuvant (pentaerythritol) inazeotropic solvents of dioxane-water and 2-ethoxyethanol-water. A numberof Japanese Patent application Abstracts relate to spray drying ofhydrophilic-hydrophobic product combinations, including JP 806766; JP7242568; JP 7101884; JP 7101883; JP 71018982; JP 7101881; and JP4036233. Other foreign patent publications relevant to spray dryinghydrophilic-hydrophobic product combinations include FR 2594693; DE2209477; and WO 88/07870.

Lyophilization.

An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g.,a precursor, e.g., a larger iRNA agent which can be processed into asRNA agent, or a DNA which encodes an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, or precursor thereof)preparation can be made by lyophilization. Lyophilization is afreeze-drying process in which water is sublimed from the compositionafter it is frozen. The particular advantage associated with thelyophilization process is that biologicals and pharmaceuticals that arerelatively unstable in an aqueous solution can be dried without elevatedtemperatures (thereby eliminating the adverse thermal effects), and thenstored in a dry state where there are few stability problems. Withrespect to the instant invention such techniques are particularlycompatible with the incorporation of nucleic acids in perforatedmicrostructures without compromising physiological activity. Methods forproviding lyophilized particulates are known to those of skill in theart and it would clearly not require undue experimentation to providedispersion compatible microstructures in accordance with the teachingsherein. Accordingly, to the extent that lyophilization processes may beused to provide microstructures having the desired porosity and size,they are conformance with the teachings herein and are expresslycontemplated as being within the scope of the instant invention.

Targeting

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAs. Itshould be understood, however, that these formulations, compositions andmethods can be practiced with other iRNA agents, e.g., modified iRNAagents, and such practice is within the invention.

In some embodiments, an iRNA agent, e.g., a double-stranded iRNA agent,or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which canbe processed into a sRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)is targeted to a particular cell. For example, a liposome or particle orother structure that includes a iRNA can also include a targeting moietythat recognizes a specific molecule on a target cell. The targetingmoiety can be a molecule with a specific affinity for a target cell.Targeting moieties can include antibodies directed against a proteinfound on the surface of a target cell, or the ligand or areceptor-binding portion of a ligand for a molecule found on the surfaceof a target cell. For example, the targeting moiety can recognize acancer-specific antigen of the kidney (e.g., G250, CA15-3, CA19-9, CEA,or HER2/neu) or a viral antigen, thus delivering the iRNA to a cancercell or a virus-infected cell. Exemplary targeting moieties includeantibodies (such as IgM, IgG, IgA, IgD, and the like, or a functionalportions thereof), ligands for cell surface receptors (e.g., ectodomainsthereof).

Table 3 provides a number of antigens which can be used to target aniRNA to a selected cell, such as when targeting of the iRNA agent to atissue other than the kidney is desired.

TABLE 3 Targeting Antigens ANTIGEN Exemplary tumor tissue CEA(carcinoembryonic antigen) colon, breast, lung PSA (prostate specificantigen) prostate cancer CA-125 ovarian cancer CA 15-3 breast cancer CA19-9 breast cancer HER2/neu breast cancer α-feto protein testicularcancer, hepatic cancer β-HCG (human chorionic gonadotropin) testicularcancer, choriocarcinoma MUC-1 breast cancer Estrogen receptor breastcancer, uterine cancer Progesterone receptor breast cancer, uterinecancer EGFr (epidermal growth factor receptor) bladder cancer

In one embodiment, the targeting moiety is attached to a liposome. Forexample, U.S. Pat. No. 6,245,427 describes a method for targeting aliposome using a protein or peptide. In another example, a cationiclipid component of the liposome is derivatized with a targeting moiety.For example, WO 96/37194 describes convertingN-glutaryldioleoylphosphatidyl ethanolamine to a N-hydroxysuccinimideactivated ester. The product was then coupled to an RGD peptide.

Genes and Diseases

In one aspect, the invention features, a method of treating a subject atrisk for or afflicted with unwanted cell proliferation, e.g., malignantor nonmalignant cell proliferation. The method includes:

providing an iRNA agent, e.g., an sRNA or iRNA agent described herein,e.g., an iRNA having a structure described herein, where the iRNA ishomologous to and can silence, e.g., by cleavage, a gene which promotesunwanted cell proliferation;

administering an iRNA agent, e.g., an sRNA or iRNA agent describedherein to a subject, preferably a human subject,

thereby treating the subject.

In a preferred embodiment the gene is a growth factor or growth factorreceptor gene, a kinase, e.g., a protein tyrosine, serine or threoninekinase gene, an adaptor protein gene, a gene encoding a G proteinsuperfamily molecule, or a gene encoding a transcription factor.

In a preferred embodiment the iRNA agent silences the PDGF beta gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted PDGF beta expression, e.g., testicular andlung cancers.

In another preferred embodiment the iRNA agent silences the Erb-B gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted Erb-B expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences the Src gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted Src expression, e.g., colon cancers.

In a preferred embodiment the iRNA agent silences the CRK gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted CRK expression, e.g., colon and lung cancers.

In a preferred embodiment the iRNA agent silences the GRB2 gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted GRB2 expression, e.g., squamous cellcarcinoma.

In another preferred embodiment the iRNA agent silences the RAS gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted RAS expression, e.g., pancreatic, colon andlung cancers, and chronic leukemia.

In another preferred embodiment the iRNA agent silences the MEKK gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted MEKK expression, e.g., squamous cellcarcinoma, melanoma or leukemia.

In another preferred embodiment the iRNA agent silences theJNK gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted JNK expression, e.g., pancreatic or breastcancers.

In a preferred embodiment the iRNA agent silences the RAF gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted RAF expression, e.g., lung cancer or leukemia.

In a preferred embodiment the iRNA agent silences the Erk½ gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted Erk½ expression, e.g., lung cancer.

In another preferred embodiment the iRNA agent silences the PCNA(p21)gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted PCNA expression, e.g., lung cancer.

In a preferred embodiment the iRNA agent silences the MYB gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted MYB expression, e.g., colon cancer or chronicmyelogenous leukemia.

In a preferred embodiment the iRNA agent silences the c-MYC gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted c-MYC expression, e.g., Burkitt's lymphoma orneuroblastoma.

In another preferred embodiment the iRNA agent silences the JUN gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted JUN expression, e.g., ovarian, prostate orbreast cancers.

In another preferred embodiment the iRNA agent silences the FOS gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted FOS expression, e.g., skin or prostatecancers.

In a preferred embodiment the iRNA agent silences the BCL-2 gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted BCL-2 expression, e.g., lung or prostatecancers or Non-Hodgkin lymphoma.

In a preferred embodiment the iRNA agent silences the Cyclin D gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted Cyclin D expression, e.g., esophageal andcolon cancers.

In a preferred embodiment the iRNA agent silences the VEGF gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted VEGF expression, e.g., esophageal and coloncancers.

In a preferred embodiment the iRNA agent silences the EGFR gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted EGFR expression, e.g., breast cancer.

In another preferred embodiment the iRNA agent silences the Cyclin Agene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted Cyclin A expression, e.g., lung andcervical cancers.

In another preferred embodiment the iRNA agent silences the Cyclin Egene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted Cyclin E expression, e.g., lung andbreast cancers.

In another preferred embodiment the iRNA agent silences the WNT-1 gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted WNT-1 expression, e.g., basal cell carcinoma.

In another preferred embodiment the iRNA agent silences the beta-cateningene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted beta-catenin expression, e.g.,adenocarcinoma or hepatocellular carcinoma.

In another preferred embodiment the iRNA agent silences the c-MET gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted c-MET expression, e.g., hepatocellularcarcinoma.

In another preferred embodiment the iRNA agent silences the PKC gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted PKC expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences the NFKB gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted NFKB expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences the STAT3 gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted STAT3 expression, e.g., prostate cancer.

In another preferred embodiment the iRNA agent silences the survivingene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted surviving expression, e.g., cervicalor pancreatic cancers.

In another preferred embodiment the iRNA agent silences the Her2/Neugene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted Her2/Neu expression, e.g., breastcancer.

In another preferred embodiment the iRNA agent silences thetopoisomerase I gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted topoisomerase Iexpression, e.g., ovarian and colon cancers.

In a preferred embodiment the iRNA agent silences the topoisomerase IIalpha gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted topoisomerase II expression,e.g., breast and colon cancers.

In a preferred embodiment the iRNA agent silences mutations in the p73gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted p73 expression, e.g., colorectaladenocarcinoma.

In a preferred embodiment the iRNA agent silences mutations in thep21(WAF1/CIP1) gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted p21(WAF1/CIP1)expression, e.g., liver cancer.

In a preferred embodiment the iRNA agent silences mutations in thep27(KIP1) gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted p27(KIP1) expression,e.g., liver cancer.

In a preferred embodiment the iRNA agent silences mutations in the PPM1Dgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted PPM1D expression, e.g., breastcancer.

In a preferred embodiment the iRNA agent silences mutations in the RASgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted RAS expression, e.g., breast cancer.

In another preferred embodiment the iRNA agent silences mutations in thecaveolin I gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted caveolin I expression,e.g., esophageal squamous cell carcinoma.

In another preferred embodiment the iRNA agent silences mutations in theMIB I gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted MIB I expression, e.g., malebreast carcinoma (MBC).

In another preferred embodiment the iRNA agent silences mutations in theMTAI gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted MTAI expression, e.g., ovariancarcinoma.

In another preferred embodiment the iRNA agent silences mutations in theM68 gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted M68 expression, e.g., humanadenocarcinomas of the esophagus, stomach, colon, and rectum.

In preferred embodiments the iRNA agent silences mutations in tumorsuppressor genes, and thus can be used as a method to promote apoptoticactivity in combination with chemotherapeutics.

In a preferred embodiment the iRNA agent silences mutations in the p53tumor suppressor gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted p53 expression, e.g.,gall bladder, pancreatic and lung cancers.

In a preferred embodiment the iRNA agent silences mutations in the p53family member DN-p63, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted DN-p63 expression,e.g., squamous cell carcinoma In a preferred embodiment the iRNA agentsilences mutations in the pRb tumor suppressor gene, and thus can beused to treat a subject having or at risk for a disorder characterizedby unwanted pRb expression, e.g., oral squamous cell carcinoma

In a preferred embodiment the iRNA agent silences mutations in the APC1tumor suppressor gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted APC1 expression, e.g.,colon cancer.

In a preferred embodiment the iRNA agent silences mutations in the BRCA1tumor suppressor gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted BRCA1 expression, e.g.,breast cancer.

In a preferred embodiment the iRNA agent silences mutations in the PTENtumor suppressor gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted PTEN expression, e.g.,hamartomas, gliomas, and prostate and endometrial cancers.

In a preferred embodiment the iRNA agent silences MLL fusion genes,e.g., MLL-AF9, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted MLL fusion gene expression,e.g., acute leukemias.

In another preferred embodiment the iRNA agent silences the BCR/ABLfusion gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted BCR/ABL fusion gene expression,e.g., acute and chronic leukemias.

In another preferred embodiment the iRNA agent silences the TEL/AML1fusion gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted TEL/AML1 fusion geneexpression, e.g., childhood acute leukemia.

In another preferred embodiment the iRNA agent silences the EWS/FLI1fusion gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted EWS/FLI1 fusion geneexpression, e.g., Ewing Sarcoma.

In another preferred embodiment the iRNA agent silences the TLS/FUS1fusion gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted TLS/FUS1 fusion geneexpression, e.g., Myxoid liposarcoma.

In another preferred embodiment the iRNA agent silences the PAX3/FKHRfusion gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted PAX3/FKHR fusion geneexpression, e.g., Myxoid liposarcoma.

In another preferred embodiment the iRNA agent silences the AML1/ETOfusion gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted AML1/ETO fusion geneexpression, e.g., acute leukemia.

In another aspect, the invention features, a method of treating asubject, e.g., a human, at risk for or afflicted with a disease ordisorder that may benefit by angiogenesis inhibition e.g., cancer. Themethod includes:

providing an iRNA agent, e.g., an iRNA agent having a structuredescribed herein, which iRNA agent is homologous to and can silence,e.g., by cleavage, a gene which mediates angiogenesis;

administering the iRNA agent to a subject, thereby treating the subject.

In a preferred embodiment the iRNA agent silences the alpha v-integringene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted alpha V integrin, e.g., brain tumorsor tumors of epithelial origin.

In a preferred embodiment the iRNA agent silences the Flt-1 receptorgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted Flt-1 receptors, eg. Cancer andrheumatoid arthritis.

In a preferred embodiment the iRNA agent silences the tubulin gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted tubulin, eg. Cancer and retinalneovascularization.

In a preferred embodiment the iRNA agent silences the tubulin gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted tubulin, eg. Cancer and retinalneovascularization.

In another aspect, the invention features a method of treating a subjectinfected with a virus or at risk for or afflicted with a disorder ordisease associated with a viral infection. The method includes:

providing an iRNA agent, e.g., and iRNA agent having a structuredescribed herein, which iRNA agent is homologous to and can silence,e.g., by cleavage, a viral gene of a cellular gene which mediates viralfunction, e.g., entry or growth;

administering the iRNA agent to a subject, preferably a human subject,

thereby treating the subject.

Thus, the invention provides for a method of treating patients infectedby the Human Papilloma Virus (HPV) or at risk for or afflicted with adisorder mediated by HPV, e.g, cervical cancer. HPV is linked to 95% ofcervical carcinomas and thus an antiviral therapy is an attractivemethod to treat these cancers and other symptoms of viral infection.

In a preferred embodiment, the expression of a HPV gene is reduced. Inanother preferred embodiment, the HPV gene is one of the group of E2,E6, or E7.

In a preferred embodiment the expression of a human gene that isrequired for HPV replication is reduced.

The invention also includes a method of treating patients infected bythe Human Immunodeficiency Virus (HIV) or at risk for or afflicted witha disorder mediated by HIV, e.g., Acquired Immune Deficiency Syndrome(AIDS).

In a preferred embodiment, the expression of a HIV gene is reduced. Inanother preferred embodiment, the HIV gene is CCR5, Gag, or Rev.

In a preferred embodiment the expression of a human gene that isrequired for HIV replication is reduced. In another preferredembodiment, the gene is CD4 or Tsg101.

The invention also includes a method for treating patients infected bythe Hepatitis B Virus (HBV) or at risk for or afflicted with a disordermediated by HBV, e.g., cirrhosis and heptocellular carcinoma.

In a preferred embodiment, the expression of a HBV gene is reduced. Inanother preferred embodiment, the targeted HBV gene encodes one of thegroup of the tail region of the HBV core protein, the pre-cregious(pre-c) region, or the cregious (c) region. In another preferredembodiment, a targeted HBV—RNA sequence is comprised of the poly(A)tail.

In preferred embodiment the expression of a human gene that is requiredfor HBV replication is reduced.

The invention also provides for a method of treating patients infectedby the Hepatitis A Virus (HAV), or at risk for or afflicted with adisorder mediated by HAV.

In a preferred embodiment the expression of a human gene that isrequired for HAV replication is reduced.

The present invention provides for a method of treating patientsinfected by the Hepatitis C Virus (HCV), or at risk for or afflictedwith a disorder mediated by HCV, e.g., cirrhosis

In a preferred embodiment, the expression of a HCV gene is reduced.

In another preferred embodiment the expression of a human gene that isrequired for HCV replication is reduced.

The present invention also provides for a method of treating patientsinfected by the any of the group of Hepatitis Viral strains comprisinghepatitis D, E, F, G, or H, or patients at risk for or afflicted with adisorder mediated by any of these strains of hepatitis.

In a preferred embodiment, the expression of a Hepatitis, D, E, F, G, orH gene is reduced.

In another preferred embodiment the expression of a human gene that isrequired for hepatitis D, E, F, G or H replication is reduced.

Methods of the invention also provide for treating patients infected bythe Respiratory Syncytial Virus (RSV) or at risk for or afflicted with adisorder mediated by RSV, e.g, lower respiratory tract infection ininfants and childhood asthma, pneumonia and other complications, e.g.,in the elderly.

In a preferred embodiment, the expression of a RSV gene is reduced. Inanother preferred embodiment, the targeted HBV gene encodes one of thegroup of genes N, L, or P.

In a preferred embodiment the expression of a human gene that isrequired for RSV replication is reduced.

Methods of the invention provide for treating patients infected by theHerpes Simplex Virus (HSV) or at risk for or afflicted with a disordermediated by HSV, e.g, genital herpes and cold sores as well aslife-threatening or sight-impairing disease mainly in immunocompromisedpatients.

In a preferred embodiment, the expression of a HSV gene is reduced. Inanother preferred embodiment, the targeted HSV gene encodes DNApolymerase or the helicase-primase.

In a preferred embodiment the expression of a human gene that isrequired for HSV replication is reduced.

The invention also provides a method for treating patients infected bythe herpes Cytomegalovirus (CMV) or at risk for or afflicted with adisorder mediated by CMV, e.g., congenital virus infections andmorbidity in immunocompromised patients.

In a preferred embodiment, the expression of a CMV gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for CMV replication is reduced.

Methods of the invention also provide for a method of treating patientsinfected by the herpes Epstein Barr Virus (EBV) or at risk for orafflicted with a disorder mediated by EBV, e.g., NK/T-cell lymphoma,non-Hodgkin lymphoma, and Hodgkin disease.

In a preferred embodiment, the expression of a EBV gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for EBV replication is reduced.

Methods of the invention also provide for treating patients infected byKaposi's Sarcoma-associated Herpes Virus (KSHV), also called humanherpesvirus 8, or patients at risk for or afflicted with a disordermediated by KSHV, e.g., Kaposi's sarcoma, multicentric Castleman'sdisease and AIDS-associated primary effusion lymphoma.

In a preferred embodiment, the expression of a KSHV gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for KSHV replication is reduced.

The invention also includes a method for treating patients infected bythe JC Virus (JCV) or a disease or disorder associated with this virus,e.g., progressive multifocal leukoencephalopathy (PML).

In a preferred embodiment, the expression of a JCV gene is reduced.

In preferred embodiment the expression of a human gene that is requiredfor JCV replication is reduced.

Methods of the invention also provide for treating patients infected bythe myxovirus or at risk for or afflicted with a disorder mediated bymyxovirus, e.g., influenza.

In a preferred embodiment, the expression of a myxovirus gene isreduced.

In a preferred embodiment the expression of a human gene that isrequired for myxovirus replication is reduced.

Methods of the invention also provide for treating patients infected bythe rhinovirus or at risk for of afflicted with a disorder mediated byrhinovirus, e.g., the common cold.

In a preferred embodiment, the expression of a rhinovirus gene isreduced.

In preferred embodiment the expression of a human gene that is requiredfor rhinovirus replication is reduced.

Methods of the invention also provide for treating patients infected bythe coronavirus or at risk for of afflicted with a disorder mediated bycoronavirus, e.g., the common cold.

In a preferred embodiment, the expression of a coronavirus gene isreduced.

In preferred embodiment the expression of a human gene that is requiredfor coronavirus replication is reduced.

Methods of the invention also provide for treating patients infected bythe flavivirus West Nile or at risk for or afflicted with a disordermediated by West Nile Virus.

In a preferred embodiment, the expression of a West Nile Virus gene isreduced. In another preferred embodiment, the West Nile Virus gene isone of the group comprising E, NS3, or NS5.

In a preferred embodiment the expression of a human gene that isrequired for West Nile Virus replication is reduced.

Methods of the invention also provide for treating patients infected bythe St. Louis

Encephalitis flavivirus, or at risk for or afflicted with a disease ordisorder associated with this virus, e.g., viral haemorrhagic fever orneurological disease.

In a preferred embodiment, the expression of a St. Louis Encephalitisgene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for St. Louis Encephalitis virus replication is reduced.

Methods of the invention also provide for treating patients infected bythe Tick-borne encephalitis flavivirus, or at risk for or afflicted witha disorder mediated by Tick-borne encephalitis virus, e.g., viralhaemorrhagic fever and neurological disease.

In a preferred embodiment, the expression of a Tick-borne encephalitisvirus gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Tick-borne encephalitis virus replication is reduced.

Methods of the invention also provide for methods of treating patientsinfected by the Murray Valley encephalitis flavivirus, which commonlyresults in viral haemorrhagic fever and neurological disease.

In a preferred embodiment, the expression of a Murray Valleyencephalitis virus gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Murray Valley encephalitis virus replication is reduced.

The invention also includes methods for treating patients infected bythe dengue flavivirus, or a disease or disorder associated with thisvirus, e.g., dengue haemorrhagic fever.

In a preferred embodiment, the expression of a dengue virus gene isreduced.

In a preferred embodiment the expression of a human gene that isrequired for dengue virus replication is reduced.

Methods of the invention also provide for treating patients infected bythe Simian Virus 40 (SV40) or at risk for or afflicted with a disordermediated by SV40, e.g., tumorigenesis.

In a preferred embodiment, the expression of a SV40 gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for SV40 replication is reduced.

The invention also includes methods for treating patients infected bythe Human T Cell Lymphotropic Virus (HTLV), or a disease or disorderassociated with this virus, e.g., leukemia and myelopathy.

In a preferred embodiment, the expression of a HTLV gene is reduced. Inanother preferred embodiment the HTLV1 gene is the Tax transcriptionalactivator.

In a preferred embodiment the expression of a human gene that isrequired for HTLV replication is reduced.

Methods of the invention also provide for treating patients infected bythe Moloney-Murine Leukemia Virus (Mo-MuLV) or at risk for or afflictedwith a disorder mediated by Mo-MuLV, e.g., T-cell leukemia.

In a preferred embodiment, the expression of a Mo-MuLV gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Mo-MuLV replication is reduced.

Methods of the invention also provide for treating patients infected bythe encephalomyocarditis virus (EMCV) or at risk for or afflicted with adisorder mediated by EMCV, e.g. myocarditis. EMCV leads to myocarditisin mice and pigs and is capable of infecting human myocardial cells.This virus is therefore a concern for patients undergoingxenotransplantation.

In a preferred embodiment, the expression of a EMCV gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for EMCV replication is reduced.

The invention also includes a method for treating patients infected bythe measles virus (MV) or at risk for or afflicted with a disordermediated by MV, e.g. measles.

In a preferred embodiment, the expression of a MV gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for MV replication is reduced.

The invention also includes a method for treating patients infected bythe Vericella zoster virus (VZV) or at risk for or afflicted with adisorder mediated by VZV, e.g. chicken pox or shingles (also calledzoster).

In a preferred embodiment, the expression of a VZV gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for VZV replication is reduced.

The invention also includes a method for treating patients infected byan adenovirus or at risk for or afflicted with a disorder mediated by anadenovirus, e.g. respiratory tract infection.

In a preferred embodiment, the expression of an adenovirus gene isreduced.

In a preferred embodiment the expression of a human gene that isrequired for adenovirus replication is reduced.

The invention includes a method for treating patients infected by ayellow fever virus (YFV) or at risk for or afflicted with a disordermediated by a YFV, e.g. respiratory tract infection.

In a preferred embodiment, the expression of a YFV gene is reduced. Inanother preferred embodiment, the preferred gene is one of a group thatincludes the E, NS2A, or NS3 genes.

In a preferred embodiment the expression of a human gene that isrequired for YFV replication is reduced.

Methods of the invention also provide for treating patients infected bythe poliovirus or at risk for or afflicted with a disorder mediated bypoliovirus, e.g., polio.

In a preferred embodiment, the expression of a poliovirus gene isreduced.

In a preferred embodiment the expression of a human gene that isrequired for poliovirus replication is reduced.

Methods of the invention also provide for treating patients infected bya poxvirus or at risk for or afflicted with a disorder mediated by apoxvirus, e.g., smallpox

In a preferred embodiment, the expression of a poxvirus gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for poxvirus replication is reduced.

In another, aspect the invention features methods of treating a subjectinfected with a pathogen, e.g., a bacterial, amoebic, parasitic, orfungal pathogen. The method includes:

providing a iRNA agent, e.g., a siRNA having a structure describedherein, where siRNA is homologous to and can silence, e.g., by cleavageof a pathogen gene;

administering the iRNA agent to a subject, preferably a human subject,

thereby treating the subject.

The target gene can be one involved in growth, cell wall synthesis,protein synthesis, transcription, energy metabolism, e.g., the Krebscycle, or toxin production.

Thus, the present invention provides for a method of treating patientsinfected by a plasmodium that causes malaria.

In a preferred embodiment, the expression of a plasmodium gene isreduced. In another preferred embodiment, the gene is apical membraneantigen 1 (AMA1).

In a preferred embodiment the expression of a human gene that isrequired for plasmodium replication is reduced.

The invention also includes methods for treating patients infected bythe Mycobacterium ulcerans, or a disease or disorder associated withthis pathogen, e.g. Buruli ulcers.

In a preferred embodiment, the expression of a Mycobacterium ulceransgene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Mycobacterium ulcerans replication is reduced.

The invention also includes methods for treating patients infected bythe Mycobacterium tuberculosis, or a disease or disorder associated withthis pathogen, e.g. tuberculosis.

In a preferred embodiment, the expression of a Mycobacteriumtuberculosis gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Mycobacterium tuberculosis replication is reduced.

The invention also includes methods for treating patients infected bythe Mycobacterium leprae, or a disease or disorder associated with thispathogen, e.g. leprosy.

In a preferred embodiment, the expression of a Mycobacterium leprae geneis reduced.

In a preferred embodiment the expression of a human gene that isrequired for Mycobacterium leprae replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Staphylococcus aureus, or a disease or disorder associatedwith this pathogen, e.g. infections of the skin and muscous membranes.

In a preferred embodiment, the expression of a Staphylococcus aureusgene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Staphylococcus aureus replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria

Streptococcus pneumoniae, or a disease or disorder associated with thispathogen, e.g. pneumonia or childhood lower respiratory tract infection.

In a preferred embodiment, the expression of a Streptococcus pneumoniaegene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Streptococcus pneumoniae replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Streptococcus pyogenes, or a disease or disorder associatedwith this pathogen, e.g. Strep throat or Scarlet fever.

In a preferred embodiment, the expression of a Streptococcus pyogenesgene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Streptococcus pyogenes replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Chlamydia pneumoniae, or a disease or disorder associatedwith this pathogen, e.g. pneumonia or childhood lower respiratory tractinfection

In a preferred embodiment, the expression of a Chlamydia pneumoniae geneis reduced.

In a preferred embodiment the expression of a human gene that isrequired for Chlamydia pneumoniae replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Mycoplasma pneumoniae, or a disease or disorder associatedwith this pathogen, e.g. pneumonia or childhood lower respiratory tractinfection

In a preferred embodiment, the expression of a Mycoplasma pneumoniaegene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Mycoplasma pneumoniae replication is reduced.

In one aspect, the invention features, a method of treating a subject,e.g., a human, at risk for or afflicted with a disease or disordercharacterized by an unwanted immune response, e.g., an inflammatorydisease or disorder, or an autoimmune disease or disorder. The methodincludes:

providing an iRNA agent, e.g., an iRNA agent having a structuredescribed herein, which iRNA agent is homologous to and can silence,e.g., by cleavage, a gene which mediates an unwanted immune response;

administering the iRNA agent to a subject,

thereby treating the subject.

In a preferred embodiment the disease or disorder is an ischemia orreperfusion injury, e.g., ischemia or reperfusion injury associated withacute myocardial infarction, unstable angina, cardiopulmonary bypass,surgical intervention e.g., angioplasty, e.g., percutaneous transluminalcoronary angioplasty, the response to a transplantated organ or tissue,e.g., transplanted cardiac or vascular tissue; or thrombolysis.

In a preferred embodiment the disease or disorder is restenosis, e.g.,restenosis associated with surgical intervention e.g., angioplasty,e.g., percutaneous transluminal coronary angioplasty.

In a preferred embodiment the disease or disorder is Inflammatory BowelDisease, e.g., Crohn Disease or Ulcerative Colitis.

In a preferred embodiment the disease or disorder is inflammationassociated with an infection or injury.

In a preferred embodiment the disease or disorder is asthma, lupus,multiple sclerosis, diabetes, e.g., type II diabetes, arthritis, e.g.,rheumatoid or psoriatic.

In particularly preferred embodiments the iRNA agent silences anintegrin or co-ligand thereof, e.g., VLA4, VCAM, ICAM.

In particularly preferred embodiments the iRNA agent silences a selectinor co-ligand thereof, e.g., P-selectin, E-selectin (ELAM), 1-selectin,P-selectin glycoprotein-1 (PSGL-1).

In particularly preferred embodiments the iRNA agent silences acomponent of the complement system, e.g., C3, C5, C3aR, C5aR, C3convertase, C5 convertase.

In particularly preferred embodiments the iRNA agent silences achemokine or receptor thereof, e.g., TNFI, TNFJ, IL-11, IL-1J, IL-2,IL-2R, IL-4, IL-4R, IL-5, IL-6, IL-8, TNFRI, TNFRII, IgE, SCYA11, CCR3.

In other embodiments the iRNA agent silences GCSF, Gro1, Gro2, Gro3,PF4, MIG, Pro-Platelet Basic Protein (PPBP), MIP-1I, MIP-1J, RANTES,MCP-1, MCP-2, MCP-3, CMBKR1, CMBKR2, CMBKR3, CMBKR5, AIF-1, I-309.

In one aspect, the invention features, a method of treating a subject,e.g., a human, at risk for or afflicted with acute pain or chronic pain.The method includes: providing an iRNA agent, which iRNA is homologousto and can silence, e.g., by cleavage, a gene which mediates theprocessing of pain;

administering the iRNA to a subject,

thereby treating the subject.

In particularly preferred embodiments the iRNA agent silences acomponent of an ion channel.

In particularly preferred embodiments the iRNA agent silences aneurotransmitter receptor or ligand.

In one aspect, the invention features, a method of treating a subject,e.g., a human, at risk for or afflicted with a neurological disease ordisorder. The method includes:

providing an iRNA agent which iRNA is homologous to and can silence,e.g., by cleavage, a gene which mediates a neurological disease ordisorder;

administering the iRNA agent to a subject,

thereby treating the subject.

In a preferred embodiment the disease or disorder is Alzheimer's Diseaseor Parkinson Disease.

In particularly preferred embodiments the iRNA agent silences anamyloid-family gene, e.g., APP; a presenilin gene, e.g., PSEN1 andPSEN2, or I-synuclein.

In a preferred embodiment the disease or disorder is a neurodegenerativetrinucleotide repeat disorder, e.g., Huntington disease, dentatorubralpallidoluysian atrophy or a spinocerebellar ataxia, e.g., SCA1, SCA2,SCA3 (Machado-Joseph disease), SCA7 or SCA8.

In particularly preferred embodiments the iRNA agent silences HD, DRPLA,SCA1, SCA2, MJD1, CACNL1A4, SCA7, SCA8.

The loss of heterozygosity (LOH) can result in hemizygosity forsequence, e.g., genes, in the area of LOH. This can result in asignificant genetic difference between normal and disease-state cells,e.g., cancer cells, and provides a useful difference between normal anddisease-state cells, e.g., cancer cells. This difference can arisebecause a gene or other sequence is heterozygous in euploid cells but ishemizygous in cells having LOH. The regions of LOH will often include agene, the loss of which promotes unwanted proliferation, e.g., a tumorsuppressor gene, and other sequences including, e.g., other genes, insome cases a gene which is essential for normal function, e.g., growth.Methods of the invention rely, in part, on the specific cleavage orsilencing of one allele of an essential gene with an iRNA agent of theinvention. The iRNA agent is selected such that it targets the singleallele of the essential gene found in the cells having LOH but does notsilence the other allele, which is present in cells which do not showLOH. In essence, it discriminates between the two alleles,preferentially silencing the selected allele. In essence polymorphisms,e.g., SNPs of essential genes that are affected by LOH, are used as atarget for a disorder characterized by cells having LOH, e.g., cancercells having LOH.

E.g., one of ordinary skill in the art can identify essential geneswhich are in proximity to tumor suppressor genes, and which are within aLOH region which includes the tumor suppressor gene. The gene encodingthe large subunit of human RNA polymerase II, POLR2A, a gene located inclose proximity to the tumor suppressor gene p53, is such a gene. Itfrequently occurs within a region of LOH in cancer cells. Other genesthat occur within LOH regions and are lost in many cancer cell typesinclude the group comprising replication protein A 70-kDa subunit,replication protein A 32-kD, ribonucleotide reductase, thymidilatesynthase, TATA associated factor 2H, ribosomal protein S14, eukaryoticinitiation factor 5A, alanyl tRNA synthetase, cysteinyl tRNA synthetase,NaK ATPase, alpha-1 subunit, and transferrin receptor.

Accordingly, the invention features, a method of treating a disordercharacterized by LOH, e.g., cancer. The method includes:

optionally, determining the genotype of the allele of a gene in theregion of LOH and preferably determining the genotype of both alleles ofthe gene in a normal cell;

providing an iRNA agent which preferentially cleaves or silences theallele found in the LOH cells;

administerning the iRNA to the subject,

thereby treating the disorder.

The invention also includes a iRNA agent disclosed herein, e.g, an iRNAagent which can preferentially silence, e.g., cleave, one allele of apolymorphic gene In another aspect, the invention provides a method ofcleaving or silencing more than one gene with an iRNA agent. In theseembodiments the iRNA agent is selected so that it has sufficienthomology to a sequence found in more than one gene. For example, thesequence AAGCTGGCCCTGGACATGGAGAT (SEQ ID NO:28) is conserved betweenmouse lamin B1, lamin B2, keratin complex 2-gene 1 and lamin A/C. Thusan iRNA agent targeted to this sequence would effectively silence theentire collection of genes.

The invention also includes an iRNA agent disclosed herein, which cansilence more than one gene.

Route of Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention. Acomposition that includes a iRNA can be delivered to a subject by avariety of routes. Exemplary routes include: intravenous, topical,rectal, anal, vaginal, nasal, pulmonary, ocular.

The iRNA molecules of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically include one or more species of iRNA and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, or intrathecal or intraventricularadministration.

The route and site of administration may be chosen to enhance targeting.For example, to target muscle cells, intramuscular injection into themuscles of interest would be a logical choice. Lung cells might betargeted by administering the iRNA in aerosol form. The vascularendothelial cells could be targeted by coating a balloon catheter withthe iRNA and mechanically introducing the DNA.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water, syrups, elixirs or non-aqueous media,tablets, capsules, lozenges, or troches. In the case of tablets,carriers that can be used include lactose, sodium citrate and salts ofphosphoric acid. Various disintegrants such as starch, and lubricatingagents such as magnesium stearate, sodium lauryl sulfate and talc, arecommonly used in tablets. For oral administration in capsule form,useful diluents are lactose and high molecular weight polyethyleneglycols. When aqueous suspensions are required for oral use, the nucleicacid compositions can be combined with emulsifying and suspendingagents. If desired, certain sweetening and/or flavoring agents can beadded.

Compositions for intrathecal or intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

For ocular administration, ointments or droppable liquids may bedelivered by ocular delivery systems known to the art such asapplicators or eye droppers. Such compositions can include mucomimeticssuch as hyaluronic acid, chondroitin sulfate, hydroxypropylmethylcellulose or poly(vinyl alcohol), preservatives such as sorbicacid, EDTA or benzylchronium chloride, and the usual quantities ofdiluents and/or carriers.

Topical Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention. In apreferred embodiment, an iRNA agent, e.g., a double-stranded iRNA agent,or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which canbe processed into a sRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)is delivered to a subject via topical administration. “Topicaladministration” refers to the delivery to a subject by contacting theformulation directly to a surface of the subject. The most common formof topical delivery is to the skin, but a composition disclosed hereincan also be directly applied to other surfaces of the body, e.g., to theeye, a mucous membrane, to surfaces of a body cavity or to an internalsurface. As mentioned above, the most common topical delivery is to theskin. The term encompasses several routes of administration including,but not limited to, topical and transdermal. These modes ofadministration typically include penetration of the skin's permeabilitybarrier and efficient delivery to the target tissue or stratum. Topicaladministration can be used as a means to penetrate the epidermis anddermis and ultimately achieve systemic delivery of the composition.Topical administration can also be used as a means to selectivelydeliver oligonucleotides to the epidermis or dermis of a subject, or tospecific strata thereof, or to an underlying tissue.

The term “skin,” as used herein, refers to the epidermis and/or dermisof an animal. Mammalian skin consists of two major, distinct layers. Theouter layer of the skin is called the epidermis. The epidermis iscomprised of the stratum corneum, the stratum granulosum, the stratumspinosum, and the stratum basale, with the stratum corneum being at thesurface of the skin and the stratum basale being the deepest portion ofthe epidermis. The epidermis is between 50 μm and 0.2 mm thick,depending on its location on the body.

Beneath the epidermis is the dermis, which is significantly thicker thanthe epidermis. The dermis is primarily composed of collagen in the formof fibrous bundles. The collagenous bundles provide support for, interalia, blood vessels, lymph capillaries, glands, nerve endings andimmunologically active cells.

One of the major functions of the skin as an organ is to regulate theentry of substances into the body. The principal permeability barrier ofthe skin is provided by the stratum corneum, which is formed from manylayers of cells in various states of differentiation. The spaces betweencells in the stratum corneum is filled with different lipids arranged inlattice-like formations that provide seals to further enhance the skinspermeability barrier.

The permeability barrier provided by the skin is such that it is largelyimpermeable to molecules having molecular weight greater than about 750Da. For larger molecules to cross the skin's permeability barrier,mechanisms other than normal osmosis must be used.

Several factors determine the permeability of the skin to administeredagents. These factors include the characteristics of the treated skin,the characteristics of the delivery agent, interactions between both thedrug and delivery agent and the drug and skin, the dosage of the drugapplied, the form of treatment, and the post treatment regimen. Toselectively target the epidermis and dermis, it is sometimes possible toformulate a composition that comprises one or more penetration enhancersthat will enable penetration of the drug to a preselected stratum.

Transdermal delivery is a valuable route for the administration of lipidsoluble therapeutics. The dermis is more permeable than the epidermisand therefore absorption is much more rapid through abraded, burned ordenuded skin. Inflammation and other physiologic conditions thatincrease blood flow to the skin also enhance transdermal adsorption.Absorption via this route may be enhanced by the use of an oily vehicle(inunction) or through the use of one or more penetration enhancers.Other effective ways to deliver a composition disclosed herein via thetransdermal route include hydration of the skin and the use ofcontrolled release topical patches. The transdermal route provides apotentially effective means to deliver a composition disclosed hereinfor systemic and/or local therapy.

In addition, iontophoresis (transfer of ionic solutes through biologicalmembranes under the influence of an electric field) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163),phonophoresis or sonophoresis (use of ultrasound to enhance theabsorption of various therapeutic agents across biological membranes,notably the skin and the cornea) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 166), and optimization ofvehicle characteristics relative to dose position and retention at thesite of administration (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 168) may be useful methods for enhancing thetransport of topically applied compositions across skin and mucosalsites.

The compositions and methods provided may also be used to examine thefunction of various proteins and genes in vitro in cultured or preserveddermal tissues and in animals. The invention can be thus applied toexamine the function of any gene. The methods of the invention can alsobe used therapeutically or prophylactically. For example, for thetreatment of animals that are known or suspected to suffer from diseasessuch as psoriasis, lichen planus, toxic epidermal necrolysis, ertythemamultiforme, basal cell carcinoma, squamous cell carcinoma, malignantmelanoma, Paget's disease, Kaposi's sarcoma, pulmonary fibrosis, Lymedisease and viral, fungal and bacterial infections of the skin.

Pulmonary Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention. Acomposition that includes an iRNA agent, e.g., a double-stranded iRNAagent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into a sRNA agent, or a DNA which encodes an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursorthereof) can be administered to a subject by pulmonary delivery.Pulmonary delivery compositions can be delivered by inhalation by thepatient of a dispersion so that the composition, preferably iRNA, withinthe dispersion can reach the lung where it can be readily absorbedthrough the alveolar region directly into blood circulation. Pulmonarydelivery can be effective both for systemic delivery and for localizeddelivery to treat diseases of the lungs.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are preferred. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self contained. Dry powder dispersion devices, for example,deliver drugs that may be readily formulated as dry powders. A iRNAcomposition may be stably stored as lyophilized or spray-dried powdersby itself or in combination with suitable powder carriers. The deliveryof a composition for inhalation can be mediated by a dosing timingelement which can include a timer, a dose counter, time measuringdevice, or a time indicator which when incorporated into the deviceenables dose tracking, compliance monitoring, and/or dose triggering toa patient during administration of the aerosol medicament.

The term “powder” means a composition that consists of finely dispersedsolid particles that are free flowing and capable of being readilydispersed in an inhalation device and subsequently inhaled by a subjectso that the particles reach the lungs to permit penetration into thealveoli. Thus, the powder is said to be “respirable.” Preferably theaverage particle size is less than about 10 μm in diameter preferablywith a relatively uniform spheroidal shape distribution. More preferablythe diameter is less than about 7.5 μm and most preferably less thanabout 5.0 μm. Usually the particle size distribution is between about0.1 μm and about 5 μm in diameter, particularly about 0.3 μm to about 5μm.

The term “dry” means that the composition has a moisture content belowabout 10% by weight (% w) water, usually below about 5% w and preferablyless it than about 3% w. A dry composition can be such that theparticles are readily dispersible in an inhalation device to form anaerosol.

The term “therapeutically effective amount” is the amount present in thecomposition that is needed to provide the desired level of drug in thesubject to be treated to give the anticipated physiological response.

The term “physiologically effective amount” is that amount delivered toa subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carriercan be taken into the lungs with no significant adverse toxicologicaleffects on the lungs. The types of pharmaceutical excipients that areuseful as carrier include stabilizers such as human serum albumin (HSA),bulking agents such as carbohydrates, amino acids and polypeptides; pHadjusters or buffers; salts such as sodium chloride; and the like. Thesecarriers may be in a crystalline or amorphous form or may be a mixtureof the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A preferred group of carbohydratesincludes lactose, threhalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame Amino acids include alanine andglycine, with glycine being preferred.

Additives, which are minor components of the composition of thisinvention, may be included for conformational stability during spraydrying and for improving dispersibility of the powder. These additivesinclude hydrophobic amino acids such as tryptophan, tyrosine, leucine,phenylalanine, and the like.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

Pulmonary administration of a micellar iRNA formulation may be achievedthrough metered dose spray devices with propellants such astetrafluoroethane, heptafluoroethane, dimethylfluoropropane,tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFCand CFC propellants.

Oral or Nasal Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention. Boththe oral and nasal membranes offer advantages over other routes ofadministration. For example, drugs administered through these membraneshave a rapid onset of action, provide therapeutic plasma levels, avoidfirst pass effect of hepatic metabolism, and avoid exposure of the drugto the hostile gastrointestinal (GI) environment. Additional advantagesinclude easy access to the membrane sites so that the drug can beapplied, localized and removed easily.

In oral delivery, compositions can be targeted to a surface of the oralcavity, e.g., to sublingual mucosa which includes the membrane ofventral surface of the tongue and the floor of the mouth or the buccalmucosa which constitutes the lining of the cheek. The sublingual mucosais relatively permeable thus giving rapid absorption and acceptablebioavailability of many drugs. Further, the sublingual mucosa isconvenient, acceptable and easily accessible.

The ability of molecules to permeate through the oral mucosa appears tobe related to molecular size, lipid solubility and peptide proteinionization. Small molecules, less than 1000 daltons appear to crossmucosa rapidly. As molecular size increases, the permeability decreasesrapidly. Lipid soluble compounds are more permeable than non-lipidsoluble molecules. Maximum absorption occurs when molecules areun-ionized or neutral in electrical charges.

Therefore charged molecules present the biggest challenges to absorptionthrough the oral mucosae.

A pharmaceutical composition of iRNA may also be administered to thebuccal cavity of a human being by spraying into the cavity, withoutinhalation, from a metered dose spray dispenser, a mixed micellarpharmaceutical formulation as described above and a propellant. In oneembodiment, the dispenser is first shaken prior to spraying thepharmaceutical formulation and propellant into the buccal cavity.

Devices

For ease of exposition the devices, formulations, compositions andmethods in this section are discussed largely with regard to unmodifiediRNA agents. It should be understood, however, that these devices,formulations, compositions and methods can be practiced with other iRNAagents, e.g., modified iRNA agents, and such practice is within theinvention. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) canbe disposed on or in a device, e.g., a device which implanted orotherwise placed in a subject. Exemplary devices include devices whichare introduced into the vasculature, e.g., devices inserted into thelumen of a vascular tissue, or which devices themselves form a part ofthe vasculature, including stents, catheters, heart valves, and othervascular devices. These devices, e.g., catheters or stents, can beplaced in the vasculature of the lung, heart, or leg.

Other devices include non-vascular devices, e.g., devices implanted inthe peritoneum, or in organ or glandular tissue, e.g., artificialorgans. The device can release a therapeutic substance in addition to aiRNA, e.g., a device can release insulin.

Other devices include artificial joints, e.g., hip joints, and otherorthopedic implants.

In one embodiment, unit doses or measured doses of a composition thatincludes iRNA are dispensed by an implanted device. The device caninclude a sensor that monitors a parameter within a subject. Forexample, the device can include pump, e.g., and, optionally, associatedelectronics.

Tissue, e.g., cells or organs, such as the kidney, can be treated withAn iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g.,a precursor, e.g., a larger iRNA agent which can be processed into asRNA agent, or a DNA which encodes an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, or precursor thereof) ex vivoand then administered or implanted in a subject.

The tissue can be autologous, allogeneic, or xenogeneic tissue. Forexample, tissue (e.g., kidney) can be treated to reduce graft v. hostdisease. In other embodiments, the tissue is allogeneic and the tissueis treated to treat a disorder characterized by unwanted gene expressionin that tissue, such as in the kidney. In another example, tissuecontaining hematopoietic cells, e.g., bone marrow hematopoietic cells,can be treated to inhibit unwanted cell proliferation.

Introduction of treated tissue, whether autologous or transplant, can becombined with other therapies.

In some implementations, the iRNA treated cells are insulated from othercells, e.g., by a semi-permeable porous barrier that prevents the cellsfrom leaving the implant, but enables molecules from the body to reachthe cells and molecules produced by the cells to enter the body.

In one embodiment, the porous barrier is formed from alginate.

In one embodiment, a contraceptive device is coated with or contains aniRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof). Exemplary devicesinclude condoms, diaphragms, IUD (implantable uterine devices, sponges,vaginal sheaths, and birth control devices. In one embodiment, the iRNAis chosen to inactive sperm or egg. In another embodiment, the iRNA ischosen to be complementary to a viral or pathogen RNA, e.g., an RNA ofan STD. In some instances, the iRNA composition can include aspermicide.

Dosage

In one aspect, the invention features a method of administering an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, to a subject(e.g., a human subject). The method includes administering a unit doseof the iRNA agent, e.g., a sRNA agent, e.g., double stranded sRNA agentthat (a) the double-stranded part is 19-25 nucleotides (nt) long,preferably 21-23 nt, (b) is complementary to a target RNA (e.g., anendogenous or pathogen target RNA), and, optionally, (c) includes atleast one 3′ overhang 1-5 nucleotide long. In one embodiment, the unitdose is less than 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1,0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g.about 4.4×10¹⁶ copies) per kg of bodyweight, or less than 1500, 750,300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015,0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent adisease or disorder, e.g., a disease or disorder associated with thetarget RNA, such as an RNA present in the kidney. The unit dose, forexample, can be administered by injection (e.g., intravenous orintramuscular), an inhaled dose, or a topical application. Particularlypreferred dosages are less than 2, 1, or 0.1 mg/kg of body weight.

In a preferred embodiment, the unit dose is administered less frequentlythan once a day, e.g., less than every 2, 4, 8 or 30 days. In anotherembodiment, the unit dose is not administered with a frequency (e.g.,not a regular frequency). For example, the unit dose may be administereda single time.

In one embodiment, the effective dose is administered with othertraditional therapeutic modalities. In one embodiment, the subject has aviral infection and the modality is an antiviral agent other than aniRNA agent, e.g., other than a double-stranded iRNA agent, or sRNAagent. In another embodiment, the subject has atherosclerosis and theeffective dose of an iRNA agent, e.g., a double-stranded iRNA agent, orsRNA agent, is administered in combination with, e.g., after surgicalintervention, e.g., angioplasty.

In one embodiment, a subject is administered an initial dose and one ormore maintenance doses of an iRNA agent, e.g., a double-stranded iRNAagent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into a sRNA agent, or a DNA which encodes an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursorthereof). The maintenance dose or doses are generally lower than theinitial dose, e.g., one-half less of the initial dose. A maintenanceregimen can include treating the subject with a dose or doses rangingfrom 0.01 μg to 1.4 mg/kg of body weight per day, e.g., 10, 1, 0.1,0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenancedoses are preferably administered no more than once every 5, 10, or 30days. Further, the treatment regimen may last for a period of time whichwill vary depending upon the nature of the particular disease, itsseverity and the overall condition of the patient. In preferredembodiments the dosage may be delivered no more than once per day, e.g.,no more than once per 24, 36, 48, or more hours, e.g., no more than oncefor every 5 or 8 days. Following treatment, the patient can be monitoredfor changes in his condition and for alleviation of the symptoms of thedisease state. The dosage of the compound may either be increased in theevent the patient does not respond significantly to current dosagelevels, or the dose may be decreased if an alleviation of the symptomsof the disease state is observed, if the disease state has been ablated,or if undesired side-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable.

In one embodiment, the iRNA agent pharmaceutical composition includes aplurality of iRNA agent species. In another embodiment, the iRNA agentspecies has sequences that are non-overlapping and non-adjacent toanother species with respect to a naturally occurring target sequence.In another embodiment, the plurality of iRNA agent species is specificfor different naturally occurring target genes. In another embodiment,the iRNA agent is allele specific.

In some cases, a patient is treated with a iRNA agent in conjunctionwith other therapeutic modalities. For example, a patient being treatedfor a kidney disease, e.g., early stage renal disease, can beadministered an iRNA agent specific for a target gene known to enhancethe progression of the disease in conjunction with a drug known toinhibit activity of the target gene product. For example, a patient whohas early stage renal disease can be treated with an iRNA agent thattargets an SGLT2 RNA, in conjunction with the small molecule phlorizin,which is known to block sodium-glucose cotransport and to subsequentlyreduce single nephron glomerular filtration rate. In another example, apatient being treated for a cancer of the kidney can be administered aniRNA agent specific for a target essential for tumor cell proliferationin conjunction with a chemotherapy.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound of the invention is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight(see U.S. Pat. No. 6,107,094).

The concentration of the iRNA agent composition is an amount sufficientto be effective in treating or preventing a disorder or to regulate aphysiological condition in humans. The concentration or amount of iRNAagent administered will depend on the parameters determined for theagent and the method of administration, e.g. nasal, buccal, pulmonary.For example, nasal formulations tend to require much lowerconcentrations of some ingredients in order to avoid irritation orburning of the nasal passages. It is sometimes desirable to dilute anoral formulation up to 10-100 times in order to provide a suitable nasalformulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof) can include a single treatment or,preferably, can include a series of treatments. It will also beappreciated that the effective dosage of a iRNA agent such as a sRNAagent used for treatment may increase or decrease over the course of aparticular treatment. Changes in dosage may result and become apparentfrom the results of diagnostic assays as described herein. For example,the subject can be monitored after administering a iRNA agentcomposition. Based on information from the monitoring, an additionalamount of the iRNA agent composition can be administered.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual compounds, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models. In some embodiments, the animal modelsinclude transgenic animals that express a human gene, e.g. a gene thatproduces a target RNA. The transgenic animal can be deficient for thecorresponding endogenous RNA. In another embodiment, the composition fortesting includes a iRNA agent that is complementary, at least in aninternal region, to a sequence that is conserved between the target RNAin the animal model and the target RNA in a human

The inventors have discovered that iRNA agents described herein can beadministered to mammals, particularly large mammals such as nonhumanprimates or humans in a number of ways.

In one embodiment, the administration of the iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, composition is parenteral,e.g. intravenous (e.g., as a bolus or as a diffusible infusion),intradermal, intraperitoneal, intramuscular, intrathecal,intraventricular, intracranial, subcutaneous, transmucosal, buccal,sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary,intranasal, urethral or ocular. Administration can be provided by thesubject or by another person, e.g., a health care provider. Themedication can be provided in measured doses or in a dispenser whichdelivers a metered dose. Selected modes of delivery are discussed inmore detail below.

The invention provides methods, compositions, and kits, for rectaladministration or delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes a an iRNA agent,e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)described herein, e.g., a therapeutically effective amount of a iRNAagent described herein, e.g., a iRNA agent having a double strandedregion of less than 40, and preferably less than 30 nucleotides andhaving one or two 1-3 nucleotide single strand 3′ overhangs can beadministered rectally, e.g., introduced through the rectum into thelower or upper colon. This approach is particularly useful in thetreatment of, inflammatory disorders, disorders characterized byunwanted cell proliferation, e.g., polyps, or colon cancer.

The medication can be delivered to a site in the colon by introducing adispensing device, e.g., a flexible, camera-guided device similar tothat used for inspection of the colon or removal of polyps, whichincludes means for delivery of the medication.

The rectal administration of the iRNA agent is by means of an enema. TheiRNA agent of the enema can be dissolved in a saline or bufferedsolution. The rectal administration can also by means of a suppository,which can include other ingredients, e.g., an excipient, e.g., cocoabutter or hydropropylmethylcellulose.

Any of the iRNA agents described herein can be administered orally,e.g., in the form of tablets, capsules, gel capsules, lozenges, trochesor liquid syrups. Further, the composition can be applied topically to asurface of the oral cavity.

Any of the iRNA agents described herein can be administered buccally.For example, the medication can be sprayed into the buccal cavity orapplied directly, e.g., in a liquid, solid, or gel form to a surface inthe buccal cavity. This administration is particularly desirable for thetreatment of inflammations of the buccal cavity, e.g., the gums ortongue, e.g., in one embodiment, the buccal administration is byspraying into the cavity, e.g., without inhalation, from a dispenser,e.g., a metered dose spray dispenser that dispenses the pharmaceuticalcomposition and a propellant.

Any of the iRNA agents described herein can be administered to oculartissue. For example, the medications can be applied to the surface ofthe eye or nearby tissue, e.g., the inside of the eyelid. They can beapplied topically, e.g., by spraying, in drops, as an eyewash, or anointment. Administration can be provided by the subject or by anotherperson, e.g., a health care provider. The medication can be provided inmeasured doses or in a dispenser which delivers a metered dose. Themedication can also be administered to the interior of the eye, and canbe introduced by a needle or other delivery device which can introduceit to a selected area or structure. Ocular treatment is particularlydesirable for treating inflammation of the eye or nearby tissue.

Any of the iRNA agents described herein can be administered directly tothe skin. For example, the medication can be applied topically ordelivered in a layer of the skin, e.g., by the use of a microneedle or abattery of microneedles which penetrate into the skin, but preferablynot into the underlying muscle tissue. Administration of the iRNA agentcomposition can be topical. Topical applications can, for example,deliver the composition to the dermis or epidermis of a subject. Topicaladministration can be in the form of transdermal patches, ointments,lotions, creams, gels, drops, suppositories, sprays, liquids or powders.A composition for topical administration can be formulated as aliposome, micelle, emulsion, or other lipophilic molecular assembly. Thetransdermal administration can be applied with at least one penetrationenhancer, such as iontophoresis, phonophoresis, and sonophoresis.

Any of the iRNA agents described herein can be administered to thepulmonary system. Pulmonary administration can be achieved by inhalationor by the introduction of a delivery device into the pulmonary system,e.g., by introducing a delivery device which can dispense themedication. A preferred method of pulmonary delivery is by inhalation.The medication can be provided in a dispenser which delivers themedication, e.g., wet or dry, in a form sufficiently small such that itcan be inhaled. The device can deliver a metered dose of medication. Thesubject, or another person, can administer the medication.

Pulmonary delivery is effective not only for disorders which directlyaffect pulmonary tissue, but also for disorders which affect othertissue.

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder,crystal, or aerosol for pulmonary delivery.

Any of the iRNA agents described herein can be administered nasally.Nasal administration can be achieved by introduction of a deliverydevice into the nose, e.g., by introducing a delivery device which candispense the medication. Methods of nasal delivery include spray,aerosol, liquid, e.g., by drops, or by topical administration to asurface of the nasal cavity. The medication can be provided in adispenser with delivery of the medication, e.g., wet or dry, in a formsufficiently small such that it can be inhaled. The device can deliver ametered dose of medication. The subject, or another person, canadminister the medication.

Nasal delivery is effective not only for disorders which directly affectnasal tissue, but also for disorders which affect other tissue

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder,crystal, or for nasal delivery.

An iRNA agent can be packaged in a viral natural capsid or in achemically or enzymatically produced artificial capsid or structurederived therefrom.

The dosage of a pharmaceutical composition including a iRNA agent can beadministered in order to alleviate the symptoms of a disease state,e.g., cancer or a cardiovascular disease. A subject can be treated withthe pharmaceutical composition by any of the methods mentioned above.

Gene expression in a subject can be modulated by administering apharmaceutical composition including an iRNA agent.

A subject can be treated by administering a defined amount of an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent) composition that is in a powdered form, e.g., a collection ofmicroparticles, such as crystalline particles. The composition caninclude a plurality of iRNA agents, e.g., specific for one or moredifferent endogenous target RNAs. The method can include other featuresdescribed herein.

A subject can be treated by administering a defined amount of an iRNAagent composition that is prepared by a method that includesspray-drying, i.e. atomizing a liquid solution, emulsion, or suspension,immediately exposing the droplets to a drying gas, and collecting theresulting porous powder particles. The composition can include aplurality of iRNA agents, e.g., specific for one or more differentendogenous target RNAs. The method can include other features describedherein.

The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent,(e.g., a precursor, e.g., a larger iRNA agent which can be processedinto a sRNA agent, or a DNA which encodes an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, or precursor thereof), can beprovided in a powdered, crystallized or other finely divided form, withor without a carrier, e.g., a micro- or nano-particle suitable forinhalation or other pulmonary delivery. This can include providing anaerosol preparation, e.g., an aerosolized spray-dried composition. Theaerosol composition can be provided in and/or dispensed by a metereddose delivery device.

The subject can be treated for a condition treatable by inhalation,e.g., by aerosolizing a spray-dried iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into a sRNA agent, or a DNA which encodes an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursorthereof) composition and inhaling the aerosolized composition. The iRNAagent can be an sRNA. The composition can include a plurality of iRNAagents, e.g., specific for one or more different endogenous target RNAs.The method can include other features described herein.

A subject can be treated by, for example, administering a compositionincluding an effective/defined amount of an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof), wherein the composition is prepared by amethod that includes spray-drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques

In another aspect, the invention features a method that includes:evaluating a parameter related to the abundance of a transcript in acell of a subject; comparing the evaluated parameter to a referencevalue; and if the evaluated parameter has a preselected relationship tothe reference value (e.g., it is greater), administering a iRNA agent(or a precursor, e.g., a larger iRNA agent which can be processed into asRNA agent, or a DNA which encodes a iRNA agent or precursor thereof) tothe subject. In one embodiment, the iRNA agent includes a sequence thatis complementary to the evaluated transcript. For example, the parametercan be a direct measure of transcript levels, a measure of a proteinlevel, a disease or disorder symptom or characterization (e.g., rate ofcell proliferation and/or tumor mass, viral load).

In another aspect, the invention features a method that includes:administering a first amount of a composition that comprises an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof) to a subject, whereinthe iRNA agent includes a strand substantially complementary to a targetnucleic acid; evaluating an activity associated with a protein encodedby the target nucleic acid; wherein the evaluation is used to determineif a second amount should be administered. In a preferred embodiment themethod includes administering a second amount of the composition,wherein the timing of administration or dosage of the second amount is afunction of the evaluating. The method can include other featuresdescribed herein.

In another aspect, the invention features a method of administering asource of a double-stranded iRNA agent (ds iRNA agent) to a subject. Themethod includes administering or implanting a source of a ds iRNA agent,e.g., a sRNA agent, that (a) includes a double-stranded region that is19-25 nucleotides long, preferably 21-23 nucleotides, (b) iscomplementary to a target RNA (e.g., an endogenous RNA or a pathogenRNA), and, optionally, (c) includes at least one 3′ overhang 1-5 ntlong. In one embodiment, the source releases ds iRNA agent over time,e.g. the source is a controlled or a slow release source, e.g., amicroparticle that gradually releases the ds iRNA agent. In anotherembodiment, the source is a pump, e.g., a pump that includes a sensor ora pump that can release one or more unit doses.

In one aspect, the invention features a pharmaceutical composition thatincludes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof)including a nucleotide sequence complementary to a target RNA, e.g.,substantially and/or exactly complementary. The target RNA can be atranscript of an endogenous human gene. In one embodiment, the iRNAagent (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b)is complementary to an endogenous target RNA, and, optionally, (c)includes at least one 3′ overhang 1-5 nt long. In one embodiment, thepharmaceutical composition can be an emulsion, microemulsion, cream,jelly, or liposome.

In one example the pharmaceutical composition includes an iRNA agentmixed with a topical delivery agent. The topical delivery agent can be aplurality of microscopic vesicles. The microscopic vesicles can beliposomes. In a preferred embodiment the liposomes are cationicliposomes.

In another aspect, the pharmaceutical composition includes an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof) admixed with a topicalpenetration enhancer. In one embodiment, the topical penetrationenhancer is a fatty acid. The fatty acid can be arachidonic acid, oleicacid, lauric acid, caprylic acid, capric acid, myristic acid, palmiticacid, stearic acid, linoleic acid, linolenic acid, dicaprate,tricaprate, monolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester, monoglyceride, diglyceride or pharmaceuticallyacceptable salt thereof.

In another embodiment, the topical penetration enhancer is a bile salt.The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid,glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid,taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid,sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate,polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable saltthereof.

In another embodiment, the penetration enhancer is a chelating agent.The chelating agent can be EDTA, citric acid, a salicyclate, a N-acylderivative of collagen, laureth-9, an N-amino acyl derivative of abeta-diketone or a mixture thereof.

In another embodiment, the penetration enhancer is a surfactant, e.g.,an ionic or nonionic surfactant. The surfactant can be sodium laurylsulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether,a perfluorchemical emulsion or mixture thereof.

In another embodiment, the penetration enhancer can be selected from agroup consisting of unsaturated cyclic ureas, 1-alkyl-alkones,1-alkenylazacyclo-alakanones, steroidal anti-inflammatory agents andmixtures thereof. In yet another embodiment the penetration enhancer canbe a glycol, a pyrrol, an azone, or a terpenes.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in aform suitable for oral delivery. In one embodiment, oral delivery can beused to deliver an iRNA agent composition to a cell or a region of thegastro-intestinal tract, e.g., small intestine, colon (e.g., to treat acolon cancer), and so forth. The oral delivery form can be tablets,capsules or gel capsules. In one embodiment, the iRNA agent of thepharmaceutical composition modulates expression of a cellular adhesionprotein, modulates a rate of cellular proliferation, or has biologicalactivity against eukaryotic pathogens or retroviruses. In anotherembodiment, the pharmaceutical composition includes an enteric materialthat substantially prevents dissolution of the tablets, capsules or gelcapsules in a mammalian stomach. In a preferred embodiment the entericmaterial is a coating. The coating can be acetate phthalate, propyleneglycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate or cellulose acetate phthalate.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes a penetration enhancer. The penetration enhancercan be a bile salt or a fatty acid. The bile salt can be ursodeoxycholicacid, chenodeoxycholic acid, and salts thereof. The fatty acid can becapric acid, lauric acid, and salts thereof.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes an excipient. In one example the excipient ispolyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes a plasticizer. The plasticizer can be diethylphthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethylcitrate.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent and a delivery vehicle. In one embodiment, theiRNA agent is (a) is 19-25 nucleotides long, preferably 21-23nucleotides, (b) is complementary to an endogenous target RNA, and,optionally, (c) includes at least one 3′ overhang 1-5 nucleotides long.

In one embodiment, the delivery vehicle can deliver an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof) to a cell by a topical route ofadministration. The delivery vehicle can be microscopic vesicles. In oneexample the microscopic vesicles are liposomes. In a preferredembodiment the liposomes are cationic liposomes. In another example themicroscopic vesicles are micelles. In one aspect, the invention featuresa pharmaceutical composition including an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof) in an injectable dosage form. In oneembodiment, the injectable dosage form of the pharmaceutical compositionincludes sterile aqueous solutions or dispersions and sterile powders.In a preferred embodiment the sterile solution can include a diluentsuch as water; saline solution; fixed oils, polyethylene glycols,glycerin, or propylene glycol.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) inoral dosage form. In one embodiment, the oral dosage form is selectedfrom the group consisting of tablets, capsules and gel capsules. Inanother embodiment, the pharmaceutical composition includes an entericmaterial that substantially prevents dissolution of the tablets,capsules or gel capsules in a mammalian stomach. In a preferredembodiment the enteric material is a coating. The coating can be acetatephthalate, propylene glycol, sorbitan monoleate, cellulose acetatetrimellitate, hydroxy propyl methyl cellulose phthalate or celluloseacetate phthalate. In one embodiment, the oral dosage form of thepharmaceutical composition includes a penetration enhancer, e.g., apenetration enhancer described herein.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in arectal dosage form. In one embodiment, the rectal dosage form is anenema. In another embodiment, the rectal dosage form is a suppository.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in avaginal dosage form.

In one embodiment, the vaginal dosage form is a suppository. In anotherembodiment, the vaginal dosage form is a foam, cream, or gel.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in apulmonary or nasal dosage form. In one embodiment, the iRNA agent isincorporated into a particle, e.g., a macroparticle, e.g., amicrosphere. The particle can be produced by spray drying,lyophilization, evaporation, fluid bed drying, vacuum drying, or acombination thereof. The microsphere can be formulated as a suspension,a powder, or an implantable solid.

In one aspect, the invention features a spray-dried iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof) composition suitable for inhalation by asubject, including: (a) a therapeutically effective amount of a iRNAagent suitable for treating a condition in the subject by inhalation;(b) a pharmaceutically acceptable excipient selected from the groupconsisting of carbohydrates and amino acids; and (c) optionally, adispersibility-enhancing amount of a physiologically-acceptable,water-soluble polypeptide.

In one embodiment, the excipient is a carbohydrate. The carbohydrate canbe selected from the group consisting of monosaccharides, disaccharides,trisaccharides, and polysaccharides. In a preferred embodiment thecarbohydrate is a monosaccharide selected from the group consisting ofdextrose, galactose, mannitol, D-mannose, sorbitol, and sorbose. Inanother preferred embodiment the carbohydrate is a disaccharide selectedfrom the group consisting of lactose, maltose, sucrose, and trehalose.

In another embodiment, the excipient is an amino acid. In oneembodiment, the amino acid is a hydrophobic amino acid. In a preferredembodiment the hydrophobic amino acid is selected from the groupconsisting of alanine, isoleucine, leucine, methionine, phenylalanine,proline, tryptophan, and valine. In yet another embodiment the aminoacid is a polar amino acid.

In a preferred embodiment the amino acid is selected from the groupconsisting of arginine, histidine, lysine, cysteine, glycine, glutamine,serine, threonine, tyrosine, aspartic acid and glutamic acid.

In one embodiment, the dispersibility-enhancing polypeptide is selectedfrom the group consisting of human serum albumin, α-lactalbumin,trypsinogen, and polyalanine.

In one embodiment, the spray-dried iRNA agent composition includesparticles having a mass median diameter (MMD) of less than 10 microns.In another embodiment, the spray-dried iRNA agent composition includesparticles having a mass median diameter of less than 5 microns. In yetanother embodiment the spray-dried iRNA agent composition includesparticles having a mass median aerodynamic diameter (MMAD) of less than5 microns.

In certain other aspects, the invention provides kits that include asuitable container containing a pharmaceutical formulation of an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof). In certain embodimentsthe individual components of the pharmaceutical formulation may beprovided in one container. Alternatively, it may be desirable to providethe components of the pharmaceutical formulation separately in two ormore containers, e.g., one container for an iRNA agent preparation, andat least another for a carrier compound. The kit may be packaged in anumber of different configurations such as one or more containers in asingle box. The different components can be combined, e.g., according toinstructions provided with the kit. The components can be combinedaccording to a method described herein, e.g., to prepare and administera pharmaceutical composition. The kit can also include a deliverydevice.

In another aspect, the invention features a device, e.g., an implantabledevice, wherein the device can dispense or administer a composition thatincludes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof),e.g., a iRNA agent that silences an endogenous transcript. In oneembodiment, the device is coated with the composition. In anotherembodiment the iRNA agent is disposed within the device. In anotherembodiment, the device includes a mechanism to dispense a unit dose ofthe composition. In other embodiments the device releases thecomposition continuously, e.g., by diffusion. Exemplary devices includestents, catheters, pumps, artificial organs or organ components (e.g.,artificial heart, a heart valve, etc.), and sutures.

As used herein, the term “crystalline” describes a solid having thestructure or characteristics of a crystal, i.e., particles ofthree-dimensional structure in which the plane faces intersect atdefinite angles and in which there is a regular internal structure. Thecompositions of the invention may have different crystalline forms.Crystalline forms can be prepared by a variety of methods, including,for example, spray drying.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES Example 1 Diethyl2-azabutane-1,4-dicarboxylate AA

A 4.7M aqueous solution of sodium hydroxide (50 mL) was added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole)was added and the mixture was stirred at room temperature until thecompletion of reaction was ascertained by TLC (19 h). After 19 h whichit was partitioned with dichloromethane (3×100 mL). The organic layerwas dried with anhydrous sodium sulfate, filtered and evaporated. Theresidue was distilled to afford AA (28.8 g, 61%).

Example 23-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde(3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0 oC. It wasthen followed by the addition of Diethyl2-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution was brought to room temperature and stirred further for 6 h.the completion of the reaction was ascertained by TLC. The reactionmixture was concentrated in vacuum and to the ethylacetate was added toprecipitate diisopropyl urea. The suspension was filtered. The filtratewas washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate andwater. The combined organic layer was dried over sodium sulfate andconcentrated to give the crude product which was purified by columnchromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB

Example 3 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionicacid ethyl ester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidinein dimethylformamide at 0° C. The solution was continued stirring for 1h. The reaction mixture was concentrated in vacuum and the residue waterwas added and the product was extracted with ethyl acetate. The crudeproduct was purified by converting into hydrochloride salt.

Example 43-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-4H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

Hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]propionic acid ethylester AC (4.7 g, 14.8 mmol) was taken in dichloromethane. The suspensionwas cooled to 0° C. with ice. To the suspension diisopropylethylamine(3.87 g, 5.2 mL, 30 mmol) was added. To the resulting solutioncholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reactionmixture was stirred overnight. The reaction mixture was diluted withdichloromethane and washed with 10% hydrochloric acid. The product waspurified flash chromatography (10.3 g, 92%).

Example 51-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of drytoluene. The mixture was cooled to 0° C. and 5 g (6.6 mmol) of diesterwas added slowly with stirring within 20 mins. The temperature was keptbelow 5° C. during the addition. The stirring was continued for 30 minsat 0° C. and 1 mL of glacial acetic acid was added, immediately followedby 4 g of NaH₂PO₄.H₂O in 40 mL of water. The resultant mixture wasextracted with two 100 mL of dichloromethane and the combined organicextracts were washed twice with 10 mL of phosphate buffer, dried, andevaporated to dryness. The residue was dissolved in 60 mL of toluene,cooled to 0° C. and extracted with three 50 mL portions of cold pH 9.5carbonate buffer. The aqueous extracts were converted to pH 3 withphosphoric acid, and extracted with five 40 mL portions of chloroformwhich were combined, dried and evaporated to a residue. The residue waspurified by column chromatography using 25% ethylacetate/hexanes toafford 1.9 g of 13-ketoester was obtained (39%).

Example 6[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxingmixture of ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226g, 6 mmol) in tetrahydrofuran (10 mL). Stirring is continued at refluxtemperature for 1 h. After cooling to room temperature, 1 N HCl (12.5mL) was added, the mixture was extracted with ethylacetate (3×40 mL).The combined ethylacetate layer was dried over anhydrous sodium sulfateand concentrated in vacuum to yield the product which purified by columnchromatography (10% MeOH/CHCl₃). (89%).

Example 7

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-4H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added withstirring. The reaction was carried out ar room temperature forovernight. The reaction was quenched by the addition of methanol. Thereaction mixture was concentrated in vacuum and to the residuedichloromethane (50 mL) was added. The organic layer was washed with 1Maqueous sodium bicarbonate. The organic layer was dried over anhydroussodium sulfate, filtered and concentrated. The residual pyridine wasremoved by evaporating with toluene. The crude product was purified bycolumn chromatography (2% MeOH/Chloroform, R_(f)=0.5 in 5% MeOH/CHCl₃).(1.75 g, 95%)

Example 8

Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)ester AH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture was dissolved in anhydrous dichloroethane (3mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and thesolution was stirred at room temperature under argon atmosphere for 16h. It was then diluted with dichloromethane (40 mL) and washed with icecold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). Theorganic phase was dried over anhydrous sodium sulfate and concentratedto dryness. The residue was used as such for the next step.

Example 9 Cholesterol Derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) was added. The reaction mixture turned brightorange in color. The solution was agitated briefly using wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 μm/g)was added. The suspension was agitated for 2 h. The CPG was filteredthrough a sintered funnel and washed with acetonitrile, dichloromethaneand ether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The loading capacity of the CPG was measured bytaking UV measurement. (37 μM/g).

Example 10(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)phosphoramidite AJ

Compound AG (0.15 g, 0.158 mmol) was coevaporated with toluene (5 mL).To the residue N,N-tetraisopropylammonium tetrazolide (0.0089 g, 0.079mmol) was added and the mixture was dried over P₂O₅ in a vacuum oven forovernight at 40° C. The reaction mixture was dissolved in the mixture ofanhydrous acetonitrile/dichloromethane (2;1, 1 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoramidite (0.0714 g, 0.0781mL, 0.237 mmol) was added. The reaction mixture was stirred at ambienttemperature for overnight. The completion of the reaction wasascertained by TLC (1;1 ethyl acetate:hexane). The solvent was removedunder reduced pressure and the residue was dissolved in ethyl acetate(10 mL) and washed with 5% NaHCO₃ (4 mL) and brine (4 mL). The ethylacetate layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The resulting mixture was chromatographed (50:49:1,EtOAc:Hexane:triethlyamine) to afford AJ as white foam (0.152 g, 84%).

Example 11 RNA Synthesis, Deprotection and Purification Protocol

1. Synthesis:

The RNA molecules were synthesized on a 394 ABI machine using thestandard 93 step cycle written by the manufacturer with modifications toa few wait steps as described below. The solid support was controlledpore glass (CPG, lumole, 500 Å, Glen Research, Sterling Va.) and themonomers were RNA phosphoramidites with standard protecting groups(N⁶-benzoyl-5′-O-dimethoxytrityladenosine-2′tbutyldimethylsilyl-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityluridine-2′tbutyldimethylsilyl-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,N²-isobutyryl-5′-O-dimethoxytritylguanosine-2′ tbutyldimethylsilyl,3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, andN⁴-benzoyl-5′-O-dimethoxytritylcytidine-2′tbutyldimethylsilyl-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramiditefrom Chemgenes Corp MA) used at a concentration of 0.15M in acetonitrile(CH₃CN) and a coupling time of 7.5 min. The activator was thiotetrazole(0.25M), For the PO-oxidation Iodine/Water/Pyridine was used and thePS-oxidation Beaucage reagent 0.5M solution in acetomitrile was used.All reagents for synthesis were also from Glen Research.

2. Deprotection-I (Oligomer Cleavage, Base and Phosphate Deprotection)

After completion of synthesis the controlled pore glass (CPG) wastransferred to a screw cap vial (Fisher, catalog number 03-340-5N) or ascrew cap RNase free microfuge tube. The oligonucleotide was cleavedfrom the CPG with simultaneous deprotection of base and phosphate groupswith 1.0 mL of a mixture of ethanolic ammonia [ammonia:ethanol (3:1)]for 6 hours to overnight at 55° C. The vial was cooled briefly on iceand then the ethanolic ammonia mixture was transferred to a newmicrofuge tube. The CPG was washed with 3×0.25 mL portions of 50%acetonitrile (70% CH₃CN for cholesterol and such hydrophobic conjugatedoligomers). The approximate 1.75 mL of solution is best divided equallyinto two microfuge tubes, capped tightly and then cooled at −80° C. for15 min, before drying in a speed vac/lyophilizer for about 90 min.

3. Deprotection-II (Removal of 2′ TBDMS group)

The white residue obtained was resuspended in 200 μL of triethylaminetrihydrofluoride (TEA.3HF, Aldrich) and heated at 65° C. for 1.5 h toremove the tertbutyldimethylsilyl (TBDMS) groups at the 2′ position. Thereaction was then quenched with 400 μL of isopropoxytrimethylsilane(iPrOMe₃Si Aldrich) and further incubated on the heating block leavingthe caps open for 15 min; (This causes the volatileisopropxytrimethylsilylfluoride adduct to vaporize). The residualquenching reagent was removed by drying in a speed vac. The oligomer wasthen precipitated in anhydrous methanol (MeOH, 800 μL). The liquid wasremoved very carefully after spinning in a centrifuge for 5 minutes onthe highest speed available. Residual methanol was removed by dryingbriefly in a speed vac after freezing at −80° C. The crude RNA wasobtained as a white fluffy material in the microfuge tube.

4. Quantitation of Crude Oligomer or Raw Analysis

Samples were dissolved in 50% aqueous acetonitrile (0.5 mL) andquantitated as follows: Blanking was first performed with 50% aqueousacetonitrile alone (1 mL).

5 μL of sample and 995 μL of 50% acetonitrile, were mixed well in amicrofuge tube, transferred to cuvette and absorbance reading obtainedat 260 nm. The crude material is dried down and stored at −20° C.

5. Purification of Oligomers

The crude oligomers were analyzed and purified by HPLC (Mono Q PharmaciaBiotech 5/50). The buffer system is A=100 mM Tris HCl 10% HPLC gradeacetonitrile pH=8, B=100 mM Tris-HCl pH 8, 10% HPLC grade acetonitrile 1M NaCl, flow 1.0 mL/min, wavelength 260 nm. For the unmodified RNA 21mera gradient of O-0.6M NaCl is usually adequate. One can purify a smallamount of material (˜5 OD) and analyze by CGE or MS. Once the identityof this material is confirmed the crude oligomer can then be purifiedusing a larger amount of material. i.e 400D's per run, flow rate of 1mL/min and a less sensitive wavelength of 280 nm to avoid saturation ofthe detector. Fractions containing the full length oligonucleotides arethen pooled together, evaporated and finally desalted as describedbelow.

6. Desalting of Purified Oligomer

The purified dry oligomer was then desalted using either C-18 Sepakcartridges (Waters) or Sephadex G-25M (Amersham Biosciences). Thecartridge was conditioned with 10 mL each of acetonitrile, followed 50%acetonitrile, 100 mM buffer (this can be triethylammonium acetate,sodium acetate or ammonium acetate). Finally the purified oligomerdissolved thoroughly in 10 mL RNAse free water was applied to thecartridge with very slow dropwise elution. The cartridge was washed withwater (10 mL) to remove salts. And finally the salt free oligomer waseluted with 50% acetonitrile or 50% methanol directly into a screw capvial.

7. Capillary Gel Electrophoresis (CGE) and Electrospray LC/Ms

1 μL of approximately 0.04 OD oligomer is first dried down, redissolvedin water (2 μL) and then pipetted in special vials for CGE and LC/MSanalysis. In general, desalting should be carried out prior to analysis.

TABLE 4  List of RNA oligonucleotides synthesized siRNA Sequence 1S5′-CUUACGCUGAGUACUUCGAdTdT-3′ (SEQ ID NO: 29) 1AS5′-UCGAAGUACUCAGCGUAAGdTdT-3′ (SEQ ID NO: 30) 2S5′-CUUACGCUGAGUACUUCGAUU-3′ (all RNA) (SEQ ID NO: 31) 2AS5′-UCGAAGUACUCAGCGUAAGUU-3′ (all RNA) (SEQ ID NO: 32) 3S5′-CUUACGCUGAGUACUUCGAdT*dT-3′ * = PS (SEQ ID NO: 33) 3AS5′-UCGAAGUACUCAGCGUAAGdT*dT-3′ * = PS (SEQ ID NO: 34) 4S5′-C*UUACGCUGAGUACUUCGAdT*dT-3′ * = PS (SEQ ID NO: 35) 4AS5′-U*CGAAGUACUCAGCGUAAGdT*dT-3′ * = PS (SEQ ID NO: 36) 5S5′-C*UUACGCUGAGUACUUCGA*dT*dT-3′ * = PS (SEQ ID NO: 37) 5AS5′-U*CGAAGUACUCAGCGUAAGdT*dT-3′ * = PS (SEQ ID NO: 38) 6S 5′CUUACGCUGAGUACUUCGAU_(2′OMe)U_(2′OMe) 3′ (SEQ ID NO: 39) 6AS5′-UCGAAGUACUCAGCGUAAGU_(2′OMe)U_(2′OMe)-3′ (SEQ ID NO: 40) 7S 5′CUUACGCUGAGUACUUCGAU*_(2′OMe)U_(2′OMe) 3′ * = PS (SEQ ID NO: 41) 7AS5′-UCGAAGUACUCAGCGUAAGU*_(2′OMe)U_(2′OMe)-3′ * = PS (SEQ ID NO: 42) 8S5′ C*UUACGCUGAGUACUUCGAU*_(2′OMe)U_(2′OMe) 3′ * = PS (SEQ ID NO: 43) 8AS5′-U*CGAAGUACUCAGCGUAAGU*_(2′OMe)U_(2′OMe)-3′ * = PS (SEQ ID NO: 44) 9S5′-M1CUUACGCUGAGUACUUCGAdTdTM2-3′ (SEQ ID NO: 45) 9AS5′-M1UCGAAGUACUCAGCGUAAGdTdTM2-3′ (SEQ ID NO: 46) 10S5′-M1*CUUACGCUGAGUACUUCGAdTdT*M2-3′ (SEQ ID NO: 47) 10AS5′-M1*UCGAAGUACUCAGCGUAAGdTdT*M2-3′ (SEQ ID NO: 48) 11S5′-CUUACGCUGAGUACUUCGAdTdTM3-3′ (SEQ ID NO: 49) 11AS5′-UCGAAGUACUCAGCGUAAGdTdTM3-3′ (SEQ ID NO: 50) 12S5′-CUUACGCUGAGUACUUCGAdTdT*M3-3′ * = PS (SEQ ID NO: 51) 12AS5′-UCGAAGUACUCAGCGUAAGdIdT*M3-3′ * = PS (SEQ ID NO: 52) M1 = 3′-OMe-U,in which the 3′ substituent of the (U) sugar is —OCH₃. M2 = 3′-OMe-U, inwhich the 3′ substituent of the (U) sugar is —OCH₃. M3 = 3′pyrrolidinecholesterol * = PS = phosphorothioate linkage U_(2′OMe) means that the2′ substituent of the (U) sugar is —OCH₃. dT = deoxythymidine

TABLE 5 Mass data for olignucleotides in Table 4 Expected Mass siRNA(amu) LC/MS (amu) 1S 6606.09 6606.67 1AS 6693.06 6692.93 2S 6610.916610.68 2AS 6697.01 6696.782 3S 6623.03 6622.76 3AS 6709.13 6708.71 4S6639.09 4AS 6725.2 6724 5S 6655.16 5AS 6741.26 6740.56 6S 6638.966638.66 6AS 6725.06 6724.67 7S 6655.02 6654.57 7AS 6741.13 8S 6671.096670.79 8AS 6757.19 6756.84 9S 7247.29 7246.67 9AS 7333.4 7333.11 10S7263.36 10AS 7349.46 11S 7312.41 7313.06 11AS 7398.51 7397 12S 7328.487329 12AS 7414.58 7415.39

Example 12 In Vitro Activity and Cytotoxicity of Chemically ModifiedsiRNAs

Synthetic siRNAs

Firefly luciferase targeting oligoribonucleotides (antisense5′-UCGAAGUACUCUAGCGUAAGNN-3′) (SEQ ID NO:53) were synthesized andcharacterized as described above. Twelve unique sense and twelve uniqueantisense strands were mixed in all possible combinations to yield 144distinct siRNA duplexes. Sense and antisense strands were arrayed into96-well PCR plates (VWR, West Chester, Pa.) in annealing buffer (100 mMKOAc, 30 mM HEPES, 2 mM MgOAc, pH 7.4) to give a final concentration of10 μM duplex. Annealing was performed employing a thermal cycler (ABIPRISM 7000, Applied Biosystems, Foster City, Calif.) capableaccommodating the PCR plates. The plates were held at 90° C. for oneminute and 370° C. for one hour. Duplex formation was verified by nativeagarose gel electrophoresis of a random sample of the 144 sense andantisense combinations.

Cell Culture

HeLa SS6 cells were grown at 370° C. in Dulbecco's modified Eagle medium(DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mLpenicillin, and 100 □g/mL streptomycin (Invitrogen, Carlsbad, Calif.).Cells were passaged regularly to maintain exponential growth.Twenty-four hours prior to siRNA transfection, cells were seeded onopaque, white 96-well plates (Costar, Corning, N.Y.) at a concentrationof 15,000 cells/well in 150 μL antibiotic-free, phenol red-free DMEM(Invitrogen).

Dual Luciferase Gene Silencing Assays

In vitro activity of siRNAs was determined using a high-throughput96-well plate format luciferase silencing assay. Cells were firsttransiently transfected with plasmids encoding firefly (target) andrenilla (control) luciferase. DNA transfections were performed usingLipofectamine 2000 (Invitrogen) (0.5 μL/pg total DNA) and the plasmidsgWiz-Luc (Aldevron, Fargo, N. Dak.) (200 ng/well) and pRL-CMV (Promega,Madison, Wis.) (200 ng/well). After 2 h, the plasmid transfection mediumwas removed, and the firefly luciferase targeting siRNAs were added tothe cells at 100 nM concentration. siRNA transfections were performedusing TranslT-TKO (Mires, Madison, Wis.) (0.3 □L/well). After 24 h,cells were analyzed for both firefly and renilla luciferase expressionusing a plate luminometer (VICTOR, PerkinElmer, Boston, Mass.) and theDual-Glo Luciferase Assay kit (Promega). Firefly/renilla luciferaseexpression ratios were used to determine percent gene silencing relativeto mock-treated (no siRNA) controls.

Cytotoxicity Assays

Cytotoxicity assays were performed in parallel with the gene silencingassays. These assays were carried out in the exact manner as the genesilencing assays (see above) with the exception that 24 h post siRNAtranfection, cells were analyzed for cytotoxicity instead of genesilencing. Relative cell viability was determined by quantification ofcellular ATP content using the CellTiter-Glo Luminescent Cell ViabilityAssay kit (Promega).

A control and candidate iRNA agents are delineated in FIG. 15.

Relative cell viability results and activity results are representedgraphically in FIGS. 16 and 17, respectively. Essentially no activitywas observed with duplexes with 12 AS; about 50% activity was observedwith 9-11 AS; and full activity was observed with 1-8 AS.

Representative cholesterol-tethered RRMS monomers are shown in FIG. 18.An RRMS monomer having a linked solid support (bottom left) can beincorporated at the 3′ end of an RNA, e.g., an iRNA agent. An RRMSmonomer having an amidite (bottom left) can be incorporated at the 5′end or internal position of an RNA, e.g., an iRNA agent.

LCMS data for a 3′ cholesterol conjugate after PAGE purification isshown in FIG. 19.

Example 13

To evaluate the cell permeation properties of cholesterol conjugatedsiRNAs 11 sense strand containing 3′ cholesterol conjugate was annealedwith 1 antisense strand and applied to the cell culture without anytransfection agent. The 1S-1AS duplex was used as an unmodified control.Luciferase expression was silenced by the 11S-1AS duplex with a doseresponse without the transfection agent, while the unmodified duplex1S-1AS did not show any gene silencing (see FIG. 20).

Example 14

(SEQ ID NO: 54) 5′ CHOLESTEROL-CUUACGCUGAGUACUUCGAdTdT-3′

Compound 14-a (described e.g., at page 67) was used to synthesize siRNAconjugates where cholesterol was conjugated at the 5′ end of RNAmolecules. The phosphoramidite 14-a was dissolved inacetomitrile/methylene chloride 1:1 solution to give a 0.2M solution.This was used for the terminal coupling during the oligonucleotidesynthesis. For the PO-oxidation Iodine/Water/Pyridine was used and thePS-oxidation Beaucage reagent 0.5M solution in acetomitrile was used.The diamathoxy triotyl group was removed in the synthesizer and thepurification and characterization were carried out as described inexample 11.

Example 15 Additional Ligand-Conjugated Monomer Syntheses

Scheme and compound numbers refer to those recited in Example 15.

Synthesis of 4-hydroxy-L-prolinol linker

1-(6-Benzyloxycarbonylamino-hexanoyl)-4-hydroxy-pyrrolidine-2-carboxylicacid methyl ester (2a)

Referring to scheme 1,6-benzyloxyamino hexanoic acid (13.25 g, 50 mmol)was dissolved in anhydrous dichloromethane (50 mL) and cooled to 0° C.To the solution were added diisopropyl carbodiimide (6.31 g, 7.7 mL, 50mmol) and triethylamine (10.2 g, 13.7 mL, 100 mmol). After stirring for20 mins at 0° C., 4-hydroxy-L-proline methyl ester hydrochloride (9.6 g,50 mmol) was added and the stirring was continued at room temperatureunder argon for over night. The reaction mixture was evaporated todryness. To the residue ethyl acetate (100 mL) was added and thefiltered to remove diisopropyl urea. The precipitate was washed withethyl acetate (50 mL). The combined organic layer was washed with 2NHCl, saturated sodium bicarbonate and water. The organic layer was driedover sodium sulfate, filtered and evaporated to dryness. Compound 2a(R_(f)=0.6 in 10% MeOH/CHCl₃, 22 g) was obtained, which was directlyused for the next step without further purification.

[6-(4-Hydroxy-2-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid benzyl ester (3a)

To the solution of lithium borohydride (1.34 g) in anhydroustetrahydrofuran (50 mL) was added a solution of methyl ester 2a in THF(50 mL) over a period of 30 mins at 0° C. After the addition thereaction mixture was brought to room temperature and stirred furtherunder argon. The completion of the reaction was ascertained by TLC after4 h. (R_(f)=0.4 in 10% MeOH/CHCl₃). The reaction mixture was evaporatedto dryness and cooled to 0° C. To the residue 3N HCl (100 mL) was addedslowly. After stirring for 30 mins the product was extracted withdichloromethane (3×100 mL). The combined organic layer was washed withbrine and dried over sodium sulfate. Organic layer was filtered andevaporated to dryness. Compound 3a was purified by column chromatographyfirst by eluting with ethyl acetate to remove impurities followed bydichloromethane/methanol (5%) gave 14.3 g (70%)

¹H NMR (400 MHz, DMSO-d₆): Observed rotamers due to amide bond at thering. 6 7.35 (m, 5H), 5.0 (s, 2H), 4.92 (d, OH, D₂O exchangeable, 4.78(t, OH, D₂O exchangeable) 4.28 (m, 1H), 3.95 (m, 1H), 3.2-3.48 (m, 5H),2.92-3.0 (m, 2H), 2.1-2.3 (m, 2H), 1.7-2.0 (2H), 1.34-1.52 (m, 4H),1.2-1.3 (m, 2H).

¹³C NMR (100 MHz, DMSO-d₆): δ171.3, 171.1 (minor due to rotamer whichdisappears while performing at 80° C.), 156.1, 137.3, 128.3, 127.7,68.2, 65.1, 61.9, 57.5, 55.1, 36.1, 34.2, 29.3, 26.1, 25.9, 24.6, 24.1,20.77, 14.09.

(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid benzyl ester (4a)

Referring to scheme 1, compound 3a (14 g, 38.4 mmol) was co-evaporatedwith anhydrous pyridine three times and then dissolved in pyridine (60mL). To this solution dimethylamino pyridine (0.488 g, 4 mmol) andDMT-Cl (13.6 g, 40.3 mmol, 1.05 equiv.) were added at room temperature.The reaction mixture was stirred at room temperature for 16 h. Theexcess DMT-Cl was quenched by the addition of methanol (25 mL). Thesolution was dried under reduced pressure. To the residue was suspendedin ethyl acetate (300 mL) and washed with saturated bicarbonatesolution, brine and water. The organic layer was dried over anhydroussodium sulfate, filtered and evaporated. 24.2 g of the crude product wasobtained after removal of the solvent. Upon purification over silica gelusing 2% MeOH/DCM compound 4a (21.3 g, 83%) was obtained as white foamysolid.

¹H NMR (400 MHz, DMSO-d₆): δ 7.18-7.38 (m, 14H), 6.2-6.5 (m, 4H), 5.0(s, 2H), 4.9 (d, —OH, D₂O exchangeable), 4.4 (m, 1H), 4.15 (m, 1H), 3.7(s, 6H), 3.56 (m, 1H), 3.32 (m, 1H), 3.14 (m, 1H), 2.9-3.0 (m, 4H), 2.18(m, 2H), 1.8-2.1 (m, 2H), 1.1-1.5 (m, 6H).

¹³C NMR (100 MHz, CDCl₃): δ 174.7, 172.7, 171.9, 171.3, 171.2, 158.8,158.7, 158.6, 158.5, 158.4, 158.3, 156.7, 156.7, 156.6, 147.5, 145.8,145.2, 144.9, 144.7, 144.4, 139.6, 137.1, 137.04, 137.01, 136.9, 136.82,136.78, 136.55, 136.47, 136.45, 136.3, 136.28, 135.93, 135.85, 135.81,130.2, 130.1, 130.0, 129.9, 129.3, 128.69, 128.66, 128.22, 128.16,128.0, 127.99, 127.94, 127.91, 127.77, 113.52, 113.43, 113.35, 113.3,113.24, 113.19, 113.03, 86.8, 86.1, 85.9, 73.0, 71.6, 71.5, 70.5, 69.3,67.3, 67.1, 68.76, 68.71, 64.38, 63.7, 60.58, 60.0, 56.4, 55.8, 55.7,55.45, 55.41, 55.35, 55.33, 40.97, 40.87, 40.77, 37.13, 36.83, 35.13,35.00, 34.81, 34.6, 33.3, 29.8, 26.73, 25.5, 26.4, 26.2, 24.9, 24.6,24.5, 24.3, 24.2, 21.1, 14.3.

6-Amino-1-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-hexan-1-one(5)

Compound 4a (14.5 g, 21.7 mmol) was dissolved in ethyl acetate (100 mL)and purged with argon. To the solution was added 10% palladium on carbon(2 g). The flask was purged with hydrogen 2 times and stirred further atroom temperature under hydrogen atmosphere for overnight. Thedisappearance of the starting material was confirmed by the TLC. Thereaction mixture was filtered through a pad of Celite and washed withethyl acetate. The combined organic layer was concentrated under reducedpressure to afford compound 5 (10.56 g, 91%) as white solid. This wasused as such for the next step.

¹H NMR (400 MHz, DMSO-d₆): δ 7.16-7.32 (m, 9H), 6.86 (m, 4H), 5.0 (bs,1H), 4.4 (m, 1H), 3.9-4.25 (m, 2H), 3.72 (s, 6H), 3.56 (m, 1H), 3.32 (m,1H), 3.14 (m, 1H), 2.98-3.0 (m, 2H), 2.45 (m, 2H), 2.2 (m, 2H), 1.8-2.04(m, 3H), 1.1-1.45 (m, 4H).

¹³C NMR (100 MHz, DMSO-d₆): δ 17.9, 157.9, 145.1, 144.76, 135.8 135.7,129.5, 127.8, 127.7, 127.5, 126.5, 113.2, 113.1, 85.7, 85.0, 68.5, 67.4,63.3, 54.9, 41.6, 36.2, 34.2, 33.3, 32.5, 26.2, 24.7, 24.4.

Compound 4b:

The desired compound 4b is obtained from N^(Cbz)-12-aminododecanoic acid(1b) and trans-4-hydroxyproline methyl ester hydrochloride in threesteps as described for the synthesis of compound 4a from compound 1a.

Synthesis of 4-hydroxy-L-prolinol cholesterol phosphoramidite(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid10,13-dimethyl-7-octyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-4H-cyclopenta[a]phenanthren-3-yl ester (6)

Referring to scheme 2, compound 5 (13.3 g, 25 mmol) was dissolved inanhydrous dichloromethane (100 mL) and cooled to 0° C. To the solutionwere added triethylamine (7.5 g, 10 mL, 75 mmol) and cholesterylchloroformate (11.24 g, 25 mmol) successively. The reaction temperaturewas brought to ambient temperature and stirred further for 2 h. Thecompletion of the reaction was ascertained by TLC (10% MeOH/CHCl₃). Thereaction mixture was evaporated under the vacuum to afford the crudeproduct. Compound 6 (22.1 g, 93%) was obtained as a white foamy solidafter column chromatography over silica gel.

¹H NMR (400 MHz, DMSO-d₆): δ 7.12-7.3 (m, 8H), 6.95 (m, 1H), 6.84 (m,4H), 5.3 (bs, 1H), 4.92 and 4.84 (d, OH, exchangeable with D₂O),4.21-4.38 (m, 2H), 4.35 (m, 1H), 3.7 (s, 6H), 3.54 (m, 1H), 3.28 (m,2H), 3.12 (m, 1H), 2.84-2.98 (m, 3H), 2.12-2.28 (m, 3H), 1.7-2.0 (m,7H), 0.8-1.52 (m, 40H), 0.6 (s, 3H).

¹³C NMR (100 MHz, DMSO-d₆): δ 170.8, 158.0, 157.9, 155.6, 145.0, 139.7,135.8, 135.7, 129.5, 127.7, 127.5, 121.7, 113.1, 113.0, 85.7, 85.1,72.7, 68.5, 63.3, 56.1, 55.5, 54.9, 49.4, 41.8, 36.5, 35.2, 31.3, 27.7,27.3, 26.0, 24.1, 23.8, 23.2, 22.6, 22.3, 20.5, 18.9, 18.5, 11.6.

4-hydroxy-L-prolinol-cholesterol-phosphoramidite (7)

Compound 6 (4.0 g, 4.23 mmol) was coevaporated with anhydrous toluene(25 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.238 g,2.1 mmol) was added and the mixture was dried over P₂O₅ in a vacuum ovenfor overnight at 40° C. The reaction mixture was dissolved indichloromethane (25 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.9 g, 2.1 mL,6.3 mmol) was added. The reaction mixture was stirred at ambienttemperature for overnight. The completion of the reaction wasascertained by TLC(R_(f)=0.5 in 1:1 ethyl acetate:hexane). The reactionmixture was diluted with dichloromethane (50 mL) and washed with 5%NaHCO₃ (50 mL) and brine (50 mL). The organic layer was dried overanhydrous Na₂SO₄ filtered and concentrated under reduced pressure. Theresidue was purified over silica gel (50:49:1,EtOAc:Hexane:triethlyamine) to afford 7 as white foam (4.35 g, 89%).

¹H NMR (400 MHz, CDCl₃): δ 7.14-7.38 (m, 9H), 6.8 (m, 4H), 5.36 (bs,1H), 4.34-4.7 (m, 4H), 3.4-3.82 (m, 13H), 3.15 (m, 3H), 2.58 (m, 2H),1.8-2.38 (m, 12H), 0.84-1.68 (m, 49H), 0.76 (s, 3H).

³¹P NMR (161.82 MHz, CDCl₃): δ 145.9, 145.7, 145.4, 145.0 (1:2 ratio, 4peaks due to rotamers).

¹³C NMR (100 MHz, CDCl₃): δ 171.8, 158.7, 158.5, 156.3, 145.3, 144.7,140.1, 136.4, 136.36, 136.32, 135.8, 130.1, 129.2, 128.4, 128.27,128.21, 128.13, 127.9, 127.1, 126.9, 125.5, 122.6, 111.8, 117.7, 113.4,113.2, 86.16, 86.1, 74.3, 72.3, 58.5, 58.3, 58.1, 56.8, 56.3, 55.9,55.8, 55.4, 55.3, 52.2, 43.4, 43.3, 42.5, 40.8, 39.9, 39.7, 38.7, 37.2,36.7, 36.3, 36.0, 35.0, 32.1, 32.0, 30.0, 28.45, 28.4, 28.2, 26.8, 24.8,24.7, 24.69, 24.6, 24.5, 24.0, 23.0, 22.7, 21.6, 21.2, 20.6, 20.59,20.52, 19.5, 18.9, 12.0

Synthesis of Solid Support with Immobilized Cholesterol

Succinic acidmono-{5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-[6-(10,13-dimethyl-7-octyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-4H-cyclopenta[a]phenanthren-3-yloxycarbonylamino)-hexanoyl]-pyrrolidin-3-yl}ester(8):

Referring to scheme 3, Compound 6 (22 g, 23.2 mmol) was mixed withsuccinic anhydride (3.48 g, 34.8 mmol) and DMAP (0.283 g, 2.32 mmol) anddried in a vacuum at 40° C. overnight. The mixture was dissolved inanhydrous dichloromethane (50 mL), triethylamine (7 g, 9.6 mL, 70 mmol)was added and the solution was stirred at room temperature under argonatmosphere for 16 h. It was then diluted with dichloromethane (100 mL)and washed with ice cold aqueous citric acid (5% wt., 100 mL) and water(2×100 mL). The organic phase was dried over anhydrous sodium sulfateand concentrated to dryness. The crude product was purified by columnchromatography to afford compound 8 as white solid (21.7 g, 89% yield;R_(f)=0.5 in 10% MeOH/CHCl₃).

¹H NMR (400 MHz, CDCl₃): δ 7.32-7.36 (m, 2H), 7.2-7.28 (m, 7H), 6.76-6.8(m, 4H), 5.4 (bs, 1H), 4.46 (m, 2H), 3.78 (s, 6H), 3.42 (m, 1H), 3-3.18(m, 3H), 2.5-2.6 (m, 3H), 2.12-2.38 (m, 6H), 1.78-2.02 (m, 7H), 0.8-1.6(m, 42H), 0.66 (s, 3H)

¹³C NMR (100 MHz, CDCl₃): δ 176.59, 172.22, 158.78, 158.62, 145.16,139.8, 136.39, 136.22, 130.18, 130.14, 128.23, 128.0, 126.97, 122.91,113.28, 56.88, 56.32, 55.45, 55.4, 50.19, 45.47, 42.51, 39.93, 39.72,38.67, 37.14, 36.74, 36.38, 36.0, 32.1, 32.06, 28.44, 28.22, 24.5, 24.0,23.04, 22.77, 21.24, 19.55, 18.92, 12.07, 8.72

Solid Support Immobilized with Cholesterol (9)

Succinate 8 (10.45 g, 10 mmol) was dissolved in dichloroethane (50 mL).To that solution DMAP (1.22 g, 10 mmol) was added.2,2′-Dithio-bis(5-nitropyridine) (3.1 g, 10 mmol) inacetonitrile/dichloroethane (3:1, 50 mL) was added successively. To theresulting solution triphenylphosphine (2.63 g, 10 mmol) in acetonitrile(25 ml) was added. The reaction mixture turned bright orange in color.The solution was agitated briefly using wrist-action shaker (5 mins).Long chain alkyl amine-CPG (LCAA-CPG) (70 g, 155 μm/g) was added. Thesuspension was agitated for 16 h. The CPG was filtered through asintered funnel and washed with acetonitrile, dichloromethane and ethersuccessively. Unreacted amino groups were masked using aceticanhydride/pyridine. The loading capacity of the CPG was measured bytaking UV measurement. (62 μM/g).

Synthesis of 4-hydroxy-L-prolinol-rac-dioctadecy glyceryl amidite1,2-Di-O-octadecyl-rac-glycerol succinimidyl carbamate (11)

Referring to scheme 4,1,2-Di-O-octadecyl-rac-glycerol (10 g, 16.74 mmol)was dissolved in anhydrous dichloromethane (150 mL). To the solutionwere added disuccinimidyl carbonate (6.4 g, 25.1 mmol), triethylamine(10 mL) and acetonitrile (50 mL). The reaction mixture was stirred atroom temperature under argon for 6 h and then evaporated dryness. Theresidue was dissolved in dichloromethane (300 mL). It was washed withsaturated NaHCO₃ aqueous solution (3×100 mL). The organic layer wasdried over Na₂SO₄, filtered and evaporated to dryness. Compound 11 (12.8g) was obtained as colorless powder after drying in high vacuum, whichwas directly used for the next step without further purification.

(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid 2,3-bis-octadecyloxy-propyl ester (12)

Amine 5 (10.5 g, 19 7 mmol) was dissolved in anhydrous dichloromethane(50 mL) and cooled to 0° C. To the solution were added pyridine (10 mL)and compound 11 (12.5 g, 17.3 mmol) successively. The reactiontemperature was brought to ambient temperature and stirred further for 3h. The completion of the reaction was ascertained by TLC (10%MeOH/CHCl₃). The reaction mixture was diluted with dichloromethane andwashed with saturated NaHCO₃, water followed by brine. The organic layerwas dried over sodium sulfate, filtered and concentrated under vacuum toafford the crude product. Compound 12 (17.8 g, 89%) was obtained as awhite solid after column chromatography over silica gel.

¹H NMR (400 MHz, DMSO-d₆): δ 7.2-7.38 (m, 9H), 6.76 (m, 4H), 5.4 (s,3H), 4.0 (m, 2H), 3.25 (s, 6H), 2.96 (m, 2H), 2.0 (m, 3H), 3-3.18 (m,3H), 2.5-2.6 (m, 3H), 2.12-2.38 (m, 6H), 1.78-2.02 (m, 7H), 1.2-1.6 (m,76H), 0.8 (m, 3H)

¹³C NMR (100 MHz, DMSO-d₆): δ 171.89, 171.38, 158.74, 158.56, 156.70,156.6, 145.28, 144.77, 136.49, 136.33, 135.89, 135.8, 130.19, 130.15,128.25, 128.20, 128.09, 127.94, 127.13, 126.90, 113.37, 113.21, 86.68,86.05, 71.98, 70.8, 70.69, 70.61, 69.40, 65.6, 64.38, 63.8, 60.61,40.93, 38.48, 36.97, 35.0, 33.3, 32.13, 31.21, 29.9, 29.86, 29.72,29.56, 26.59, 26.30, 26.24, 24.66, 22.89, 21.26, 14.39, 14.33

4-hydroxy-L-prolinol-rac-dioctadecy glyceryl amidite (13)

Compound 12 (10.0 g, 8.65 mmol) was coevaporated with anhydrous toluene(50 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.488 g,4.32 mmol) was added and the mixture was dried over P₂O₅ in a vacuumoven for overnight at 40° C. The reaction mixture was dissolved indichloromethane (50 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (3.91 g, 4.28mL, 13 mmol) was added. The reaction mixture was stirred at ambienttemperature for overnight. The completion of the reaction wasascertained by TLC(R_(f)=0.6 in 1:1 ethyl acetate:hexane). The reactionmixture was diluted with dichloromethane (100 mL) and washed with 5%NaHCO₃ (100 mL) and brine (100 mL). The organic layer was dried overanhydrous Na₂SO₄ filtered and concentrated under reduced pressure. Theresidue was purified over silica gel (50:49:1,EtOAc:Hexane:triethlyamine) to afford 13 as white solid (15.37 g, 93%).

¹H NMR (400 MHz, CDCl₃): δ 7.16-7.38 (m, 9H), 6.78 (m, 4H), 4.62-4.78(m, 2H), 4.27 (m, 1H), 4.04-4.2 (m, 3H), 3.7-3.8 (m, 10H), 3.4-3.6 (m,11H), 3.16 (m, 4H), 2.58-2.7 (m, 4H), 2.22 (m, 3H), 2.12 (m, 1H),1.15-1.4 (m, 75H), 0.95 (m, 6H), ³¹P NMR (161.82 MHz, CDCl₃): δ 145.96,145.76, 145.45, 145.07

¹³C NMR (100 MHz, CDCl₃): δ 171.79, 171.61, 158.75, 158.58, 156.59,145.31, 144.77, 136.47, 136.35, 136.31, 135.86, 130.22, 130.19, 128.28,128.20, 128.11, 127.95, 127.15, 126.91, 113.39, 113.24, 86.11, 71.98,70.81, 70.69, 72.93, 72.2, 71.98, 70.81, 70.81, 70.69, 64.37, 63.92,58.55, 58.35, 58.36, 58.16, 59.57, 55.86, 55.44, 55.39, 46.31, 44.70,44.65, 43.36, 43.34, 41.08, 35.08, 33.45, 32.13, 30.23, 29.92, 29.88,29.72, 29.58, 26.32, 26.26, 24.85, 24.78, 24.68, 22.9, 20.58, 14.34

Synthesis of Solid Support with Immobilized Rac-Dioctadecy Glycerol (1)

Succinic acidmono-{5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-[6-(2,3-bis-octadecyloxy-propoxycarbonylamino)-hexanoyl]pyrrolidin-3-yl}ester(14)

Referring to scheme 5, Compound 12 (5.6 g, 4.8 mmol) was mixed withsuccinic anhydride (0.727 g, 7.26 mmol) and DMAP (0.062 g, 0 5 mmol) anddried in a vacuum at 40° C. overnight. The mixture was dissolved inanhydrous dichloromethane (20 mL), triethylamine (1.52 g, 2 mL, 15 mmol)was added and the solution was stirred at room temperature under argonatmosphere for 16 h. It was then diluted with dichloromethane (50 mL)and washed with ice cold aqueous citric acid (5% wt., 50 mL) and water(2×50 mL). The organic phase was dried over anhydrous sodium sulfate andconcentrated to dryness. The crude product was purified by columnchromatography to afford compound 14 as white solid (2.85 g, 47% yield;R_(f)=0.65 in 10% MeOH/CHCl₃).

¹H NMR (400 MHz, DMSO-d₆): δ 12.2 (bs, 1H), 7.18-7.4 (m, 9H), 6.82 (m,4H), 4.62-4.78 (m, 2H), 4.27 (m, 1H), 4.04-4.2 (m, 3H), 3.7-3.8 (m,10H), 3.4-3.6 (m, 11H), 3.16 (m, 4H), 2.58-2.7 (m, 4H), 2.22 (m, 3H),2.12 (m, 1H), 1.15-1.4 (m, 75H), 0.95 (m, 6H),

¹³C NMR (100 MHz, DMSO-d₆): δ 178.26, 174.23, 171.79, 171.61, 158.75,158.58, 156.59, 145.31, 144.77, 136.47, 136.35, 136.31, 135.86, 130.22,130.19, 128.28, 128.20, 128.11, 127.95, 127.15, 126.91, 113.39, 113.24,86.11, 71.98, 70.81, 70.69, 72.93, 72.2, 71.98, 70.81, 70.81, 70.69,64.37, 63.92, 58.55, 58.35, 58.36, 58.16, 59.57, 55.86, 55.44, 55.39,46.31, 44.70, 44.65, 43.36, 43.34, 41.08, 35.08, 33.45, 32.13, 30.23,29.92, 29.88, 29.72, 29.58, 28.41, 26.32, 26.26, 24.85, 24.78, 24.68,22.9, 20.58, 14.34.

Solid Support with Immobilized Rac-Dioctadecy Glycerol (15)

Succinate 14 (2 g, 1.6 mmol) was dissolved in dichloroethane (8 mL). Tothat solution DMAP (0.194 g, 1.6 mmol) was added.2,2′-Dithio-bis(5-nitropyridine) (0.496 g, 1.6 mmol) inacetonitrile/dichloroethane (3:1, 8 mL) was added successively. To theresulting solution triphenylphosphine (0.419 g, 1.6 mmol) inacetonitrile (4 ml) was added. The reaction mixture turned bright orangein color. The solution was agitated briefly using wrist-action shaker (5mins). Long chain alkyl amine-CPG (LCAA-CPG) (5.16 g, 800 μmoles, 155μm/g) was added. The suspension was agitated for 4 h. The CPG wasfiltered through a sintered funnel and washed with acetonitrile,dichloromethane and ether successively. Unreacted amino groups weremasked using acetic anhydride/pyridine. The loading capacity of the CPGwas measured by taking UV measurement. (76 μM/g).

Synthesis of 4-hydroxy-L-prolinol-vitamin E amidite

2-(2-Hexadecyl-2,5,7,8-tetramethyl-chroman-6-yloxy)-ethanol (17)

Referring to scheme 6, vitamin E (16.0 g, 37 mmol) was dissolved inacetone (100 mL). Potassium carbonate (25.5 g, 185 mmol), ethylenecarbonate (6.5 g, 75 mmol) were added to the solution. The suspensionwas stirred at reflux temperature for over night. Even though thereaction did not go to completion, the reaction mixture was concentratedin the vacuum, and the residue was taken in ethyl acetate and washedwith water. The organic layer was dried over sodium sulfate, filteredand evaporated. The crude product was purified by column chromatographyusing hexane/ethyl acetate to afford compound 17 in 65% yield (11.5 g,R_(f)=0.8 in 25% EtOAc/Hexane).

¹H NMR (400 MHz, CDCl₃): δ 4.13 (m, 2H), 3.98 (m, 2H), 2.6 (t, 2H), 2.15(s, 3H), 2.1 (s, 6H), 1.7-1.8 (m, 2H), 1.1-15 (m, 14H), 0.8-0.88 (m,12H)

¹³C NMR (100 MHz, CDCl₃): δ 145.75, 144.74, 122.83, 121.2, 118.66,117.58, 77.43, 74.74, 60.63, 40.08, 40.01, 39.59, 37.80, 37.79, 37.71,37.67, 37.6, 37.55, 37.50, 33.01, 33.0, 32.91, 31.8, 31.57, 31.69,28.20, 25.04, 25.02, 24.66, 24.01, 22.95, 22.85, 21.28, 20.98, 19.97,19.9, 19.86, 19.81, 14.42, 14.35, 12.43, 12.0, 11.5

Carbonic acid 2,5-dioxo-pyrrolidin-1-yl ester2-(2-hexadecyl-2,5,7,8-tetramethyl-chroman-6-yloxy)-ethyl ester (18)

Compound 17 (10.5 g, 22 mmol) was dissolved in anhydrous dichloromethane(150 mL). To the solution were added disuccinimidyl carbonate (8.45 g,33 mmol), triethylamine (20 mL) and acetonitrile (50 mL). The reactionmixture was stirred at room temperature under argon for over night andthen evaporated dryness. The residue was dissolved in dichloromethane(300 mL). It was washed with saturated NaHCO₃ aqueous solution (3×100mL). The organic layer was dried over Na₂SO₄, filtered and evaporated todryness. Compound 18 (10.4 g, 77%) was obtained as colorless powderafter drying in high vacuum, which was directly used for the next stepwithout further purification.

¹H NMR (400 MHz, CDCl₃): δ 7.2-7.4 (m, 4H), 3.1 (m, 1H), 2.7 (s, 4H),2.58 (t, 2H), 2.08-2.14 (m, 9H), 1.7-1.82 (m, 2H), 1-1.6 (m, 23H),0.8-0.88 (m, 12H)

¹³C NMR (100 MHz, CDCl₃): δ 168.76, 168.66, 150.6, 150.37, 141.36,138.09, 129.24, 128.44, 126.73, 125.51, 125.1, 123.74, 117.97, 77.72,75.56, 39.58, 37.71, 37.65, 37.59, 37.48, 32.98, 32.88, 31.16, 31.11,28.19, 25.83, 25.75, 25.7, 25.02, 25.01, 24.64, 24.07, 24.07, 22.93,22.84, 21.67, 21.22, 20.73, 20.38, 19.96, 19.89, 19.86, 19.82, 12.77,11.98, 11.94

(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid 2-(2-hexadecyl-2,5,7,8-tetramethyl-chroman-6-yloxy)-ethyl ester(19)

Amine 5 (8.7 g, 16 3 mmol) was dissolved in anhydrous dichloromethane(40 mL) and cooled to 0° C. To the solution were added triethylamine(5.06 g, 6.73 mL, 50 mmol) and compound 18 (10 g, 16.2 mmol)successively. The reaction temperature was brought to ambienttemperature and stirred further for 6 h. The completion of the reactionwas ascertained by TLC (10% MeOH/CHCl₃). The reaction mixture wasdiluted with dichloromethane and washed with saturated NaHCO₃, waterfollowed by brine. The organic layer was dried over sodium sulfate,filtered and concentrated under vacuum to afford the crude product.Compound 19 (14.5 g, 88%) was obtained as a white foamy solid aftercolumn chromatography over silica gel.

¹H NMR (400 MHz, DMSO-d₆): δ 7.72 (m, 1H, —NH), 7.3 (m, 4H), 7.18 (m,5H), 6.86 (m, 4H), 4.98 (s, —OH), 4.38 (m, 2H), 4.12 (m, 2H), 3.72 (s,6H), 3.56 (m, 1H), 3.22-3.32 (m, 2H), 3.16 (m, 1H), 3.0 (m, 3H), 2.2 (m,2H), 1.98 (m, 4H), 1-9 (m, 7H), 1.8 (m, 1H), 1.72 (m, 2H), 1-1.5 (m,32H), 0.82 (m, 12H)

¹³C NMR (100 MHz, DMSO-d₆): δ 171.20, 158.29, 158.16, 154.83, 148.31,145.26, 140.65, 136.04, 135.90, 135.58, 129.78, 127.97, 127.75, 127.43,126.79, 125.91, 121.58, 117.10, 113.28, 86.0, 85.32, 74.71, 68.71,63.46, 55.13, 36.91, 36.8, 36.35, 32.25, 27.75, 26.06, 24.36, 23.91,23.68, 22.68, 22.59, 19.75, 19.68, 12.74, 11.89, 11.64

4-hydroxy-L-prolinol-vitamin E amidite (20)

Compound 19 (9.2 g, 9 mmol) was coevaporated with anhydrous toluene (50mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.51 g, 4 5mmol) was added and the mixture was dried over P₂O₅ in a vacuum oven forovernight at 40° C. The reaction mixture was dissolved indichloromethane (50 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (4 g, 4.45 mL,13.5 mmol) was added. The reaction mixture was stirred at ambienttemperature for overnight. The completion of the reaction wasascertained by TLC(R_(f)=0.65 in 1:1 ethyl acetate:hexane). The reactionmixture was diluted with dichloromethane (100 mL) and washed with 5%NaHCO₃ (100 mL) and brine (100 mL). The organic layer was dried overanhydrous Na₂SO₄ filtered and concentrated under reduced pressure. Theresidue was purified over silica gel (50:49:1,EtOAc:Hexane:triethlyamine) to afford 20 as white foamy solid (10.5 g,95%).

¹H NMR (400 MHz, CDCl₃): δ 7.38 (m, 2H), 7.18-7.28 (m, 7H), 6.82 (m,4H), 5.18 (m, 1H), 4.65 (m, 2H), 4.38 (m, 2H), 4.1 (m, 1H), 3.7-3.8 (m,9H), 3.58 (m, 3H), 3.4 (m, 1H), 3.28 (m, 2H), 3.18 (m, 2H), 2.58 (m,4H), 2.26 (m, 3H), 2-2.1 (m, 10H), 1.5-1.8 (m, 10H), 1.05-1.3 (m, 32H),0.84-0.88 (m, 14H).

³¹P NMR (161.82 MHz, CDCl₃): δ 145.92, 145.78, 145.45, 145.04

¹³C NMR (100 MHz, CDCl₃): δ 172.1, 171.82, 171.63, 158.74, 158.67,155.17, 149.34, 145.31, 144.77, 140.58, 136.44, 136.34, 136.3, 135.85,135.8, 130.18, 128.25, 128.19, 128.11, 127.95, 127.82, 127.13, 126.90,126.0, 123.0, 117.87, 117.78, 117.42, 113.4, 113.29, 113.23, 86.77,86.15, 86.10, 77.42, 75.15, 72.39, 72.21, 72.01, 63.92, 58.65, 58.53,58.47, 58.35, 58.13, 56.49, 55.96, 55.85, 55.44, 55.37, 54.65, 43.44,43.31, 41.22, 40.33, 39.55, 37.74, 37.64, 37.58, 37.47, 35.06, 33.44,32.97, 32.9, 31.3, 30.1, 28.17, 26.7, 26.74, 26.68, 25.12, 24.99, 24.84,24.77, 24.64, 24.57, 24.11, 22.92, 22.83, 21.23, 21.25, 20.76, 20.63,20.56, 20.50, 19.95, 19.88, 19.84, 19.82, 19.78, 13.05, 12.20, 11.97.

Synthesis of Solid Support with Immobilized Vitamin E (22)

Succinic acidmono-(5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[2-(2-hexadecyl-2,5,7,8-tetramethyl-chroman-6-yloxy)-ethoxycarbonylamino}-hexanoyl]-pyrrolidin-3-yl)ester (21)

Referring to scheme 7, Compound 19 (5.1 g, 5 mmol) was mixed withsuccinic anhydride (0.75 g, 7.5 mmol) and DMAP (0.062 g, 0 5 mmol) anddried in a vacuum at 40° C. overnight. The mixture was dissolved inanhydrous dichloromethane (25 mL), triethylamine (1.52 g, 2 mL, 15 mmol)was added and the solution was stirred at room temperature under argonatmosphere for 16 h. It was then diluted with dichloromethane (50 mL)and washed with ice cold aqueous citric acid (5% wt., 50 mL) and water(2×50 mL). The organic phase was dried over anhydrous sodium sulfate andconcentrated to dryness. The crude product was purified by columnchromatography to afford compound 21 as white foamy solid (2.85 g, 51%yield; R_(f)=0.65 in 10% MeOH/CHCl₃).

¹H NMR (400 MHz, DMSO-d₆): δ 12.3 (bs, 1H), 7.6 (m, 1H), 7.2-7.4 (m,9H), 6.86 (m, 4H), 5.32 (m, 2H), 4.18 (m, 2H), 3.62-3.8 (s, 6H), 3.54(m, 1H), 3.42 (m, 1H), 3.34 (s, 6H), 3.21 (m, 1H), 3.0 (m, 2H), 2.46 (m,4H), 2.2 (m, 4H), 1.9 (m, 4H), 1.72 (m, 3H), 1-1.5 (m, 30H), 0.82 (m,12H).

¹³C NMR (100 MHz, DMSO-d₆): δ 173.61, 172.26, 171.17, 165.43, 159.77,158.34, 158.22, 157.02, 154.88, 153.72, 148.68, 148.36, 145.17, 144.85,143.74, 141.94, 140.63, 135.93, 129.83, 128.06, 127.77, 127.46, 125.97,121.63, 117.25, 113.35, 85.50, 74.86, 73.08, 55.19, 36.88, 32.17, 28.97,28.80, 27.58, 24.34, 23.85, 22.75, 22.66, 19.84, 19.78, 12.8, 11.94,11.72.

Solid Support with Immobilized Vitamin E (22)

Succinate 21 (2.8 g, 2.5 mmol) was dissolved in dichloroethane (12 mL).To that solution DMAP (0.306 g, 2.5 mmol) was added.2,2′-Dithio-bis(5-nitropyridine) (0.775 g, 2 5 mmol) inacetonitrile/dichloroethane (3:1, 12 mL) was added successively. To theresulting solution triphenylphosphine (0.656 g, 2.5 mmol) inacetonitrile (7 ml) was added. The reaction mixture turned bright orangein color. The solution was agitated briefly using wrist-action shaker (5mins). Long chain alkyl amine-CPG (LCAA-CPG) (8.0 g, 1240 mmoles, 155μm/g) was added. The suspension was agitated for 2 h. The CPG wasfiltered through a sintered funnel and washed with acetonitrile,dichloromethane and ether successively. Unreacted amino groups weremasked using acetic anhydride/pyridine. The loading capacity of the CPG22 was measured by taking UV measurement. (76 μM/g).

Synthesis of 4-hydroxy-L-prolinol-thicholesterol amidite (26)

N-(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-3-(pyridin-2-yldisulfanyl)-propionamide(24)

Referring to scheme 8, amine 5 (7.7 g, 14.5 mmol) was dissolved inanhydrous dichloromethane (40 mL) and cooled to 0° C. To the solutionwere added triethylamine (3.0 g, 4.2 mL, 30 mmol) and3-(Pyridin-2-yldisulfanyl)-propionic succinate ester 23 (SPDP) (4.5 g,14.4 mmol) successively. The reaction temperature was brought to ambienttemperature and stirred further for 16 h. The completion of the reactionwas ascertained by TLC (10% MeOH/CHCl₃, R_(f)=0.6). The reaction mixturewas diluted with dichloromethane and washed with saturated NaHCO₃, waterfollowed by brine. The organic layer was dried over sodium sulfate,filtered and concentrated under vacuum to afford the crude product.Compound 24 (10.58 g, 78%) was obtained as a white foamy solid aftercolumn chromatography over silica gel.

¹H NMR (400 MHz, DMSO-d₆): δ 8.45 (d, 1H), 7.9 (m, 1H), 7.8 (m, 1H),7.76 (m, 1H), 7.3 (m, 4H), 7.18 (m, 5H), 6.86 (m, 4H), 4.98 (d, —OH,1H), 4.38 (m, 1H), 4.1 (m, 1H) (s, 6H), 3.56 (m, 1H), 3.46 (m, 1H),3.21-3.34 (m, 3H), 3.14 (m, 1H), 3 (m, 2H), 2.48 (m, 2H), 2.2 (m, 2H),1.8-2.02 (m, 2H), 1.1-1.5 (4H).

¹³C NMR (100 MHz, DMSO-d₆): 6.171.32, 169.97, 159.36, 158.31, 158.18,149.80, 145.27, 138.08, 136.1, 135.9, 129.8, 128.0, 127.7, 121.4, 119.3,113.3, 85.338, 68.7, 55.3, 34.75, 34.28, 29.1, 26.3, 24.36.

4-Hydroxy-L-prohnol-thiocholesterol-DMT-alcohol 25 Compound 24 (7.5 g,10.28 mmol) was dissolved in anhydrous dichloromethane (75 mL) underargon and cooled to 0° C. To this solution were added diisopropylethylamine (2.71 g, 3.66 mL, 21 mmol) followed by thiocholesterol (4.145 g,10.28 mmol). The reaction mixture was brought to ambient temperature andstirred further for 16 h. The completion of the reaction was ascertainedby TLC (100% ethyl acetate, R_(f)=0.6). The reaction mixture wasconcentrated under reduced pressure and the residue was subjected tocolumn chromatography on silica gel. Even though there was goodseparation in hexane/ethyl acetate system, compound precipitates in thatmixture. After eluting with 4 L of ethyl acetate, the column was elutedwith 5% MeOH/dichloromethane (2 L) to obtain compound 25 as white foamysolid (8 g, 76%).

¹H NMR (400 MHz, DMSO-d₆): δ 7.88 (m, 1H), 7.3 (m, 4H), 7.17 (m, 5H),6.84 (m, 4H), 5.3 (bs, 1H), 4.89 (d, —OH), 4.38 (m, 1H), 4.1 (m, 1H),3.72 (s, 6H), 3.56 (m, 1H), 3.32 (m, 1H), 3.14 (m, 1H), 3 (m, 3H), 2.84(m, 2H), 2.64 (m, 1H), 2.42 (m, 2H), 2.2 (m, 3H), 1.8-2.0 (m, 7H),0.8-1.54 (m, 35H), 0.62 (s, 3H).

¹³C NMR (100 MHz, DMSO-d₆): δ 170.8, 158.0, 157.9, 155.6, 145.0, 139.7,135.8, 135.7, 129.5, 127.7, 127.5, 121.7, 113.1, 113.0, 85.7, 85.1,72.7, 68.5, 63.3, 60.72, 56.1, 55.5, 55.28, 54.9, 49.4, 41.8, 36.5,35.2, 31.3, 30.35, 27.7, 27.3, 26.0, 24.1, 23.8, 23.2, 22.6, 22.3,21.11, 20.5, 19.43, 18.9, 18.5, 14.4, 11.6.

4-hydroxy-L-prolinol-thiocholesterol phosphoramidite (26)

Compound 25 (5.7 g, 5.58 mmol) was coevaporated with anhydrous toluene(50 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.315 g,2.79 mmol) was added and the mixture was dried over P₂O₅ in a vacuumoven for overnight at 40° C. The reaction mixture was dissolved indichloromethane (20 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (2.48 g, 2.72mL, 8.25 mmol) was added. The reaction mixture was stirred at ambienttemperature for overnight. The completion of the reaction wasascertained by TLC(R_(f)=0.9 in ethyl acetate). The reaction mixture wasdiluted with dichloromethane (50 mL) and washed with 5% NaHCO₃ (50 mL)and brine (50 mL). The organic layer was dried over anhydrous Na₂SO₄filtered and concentrated under reduced pressure. The residue waspurified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford26 as white foamy solid (6.1 g, 89%).

¹H NMR (400 MHz, C₆D₆): δ 7.62 (m, 2H), 7.45 (m, 5H), 7.24 (m, 2H), 7.1(m, 1H), 6.82 (m, 4H), 5.64 (m, 1H), 5.38 (m, 1H), 4.7 (m, 1H), 4.54 (m,2H), 3.78 (m, 2H), 3.5 (m, 3H), 3.36 (m, 9H), 3.22 (m, 4H), 3.06 (m,3H), 2.72 (m, 1H), 2.32-2.54 (m, 5H), 1.8-2.2 (m, 10H), 1.08-1.74 (m,28H), 1.3 (m, 6H), 0.94 (m, 12H), 0.67 (s, 3H).

³¹P NMR (161.82 MHz, C₆D₆): δ 146.05, 145.91, 145.66, 145.16

¹³C NMR (100 MHz, C₆D₆): δ 171.43, 171.25, 169.87, 159.25, 159.11,146.08, 141.59, 136.66, 136.6, 130.62, 130.54, 128.63, 127.53, 127.02,121.53, 117.73, 117.57, 113.66, 113.57, 86.59, 86.54, 64.36, 58.56,58.37, 58.30, 56.96, 56.51, 56.07, 54.86, 54.77, 50.57, 50.27, 43.48,43.35, 42.55, 40.13, 39.9, 39.75, 39.56, 38.70, 36.94, 36.64, 36.29,36.19, 35.90, 34.58, 32.24, 32.08, 29.48, 29.03, 28.98, 28.6, 28.38,26.54, 24.68, 24.61, 24.54, 23.6, 23.0, 22.74, 21.26, 20.03, 19.9,19.38, 19.01, 12.06.

Synthesis of Polymer Support Immobilized with Thiocholesterol 28

4-Hydroxy-L-prolinol-thiocholesterol-succinate 27

Referring to scheme 9, Compound 25 (2.2 g, 2.15 mmol) was mixed withsuccinic anhydride (0.323 g, 3.23 mmol) and DMAP (0.026 g, 0.215 mmol)and dried in a vacuum at 40° C. overnight. The mixture was dissolved inanhydrous dichloromethane (10 mL), triethylamine (0.708 g, 0.976 mL, 7mmol) was added and the solution was stirred at room temperature underargon atmosphere for 16 h. It was then diluted with dichloromethane (50mL) and washed with ice cold aqueous citric acid (5% wt., 25 mL) andwater (2×25 mL). The organic phase was dried over anhydrous sodiumsulfate and concentrated to dryness. The crude product was purified bycolumn chromatography to afford compound 27 as white foamy solid (2.2 g,92% yield; R_(f)=0.6s in 10% MeOH/CHCl₃).

¹H NMR (400 MHz, DMSO-d₆): δ 12.22 (bs, 1H), 7.84 (m, 1H), 7.25 (m, 4H),7.2 (m, 5H), 6.86 (m, 4H), 5.36 (m, 2H), 4.18 (bs, 1H), 3.72 (s, 6H),3.4-3.6 (m, 2H), 3.2 (m, 1H), 3.0 (m, 4H), 2.84 (m, 2H), 2.64 (m, 2H),2.4-2.52 (m, 12H), 2.2 (m, 6H), 1.9 (m, 8H), 0.8-1.52 (m, 28H), 0.65 (s,3H).

¹³C NMR (100 MHz, DMSO-d₆): δ 173.35, 171.94, 170.63, 169.64, 157.99,144.96, 141.02, 135.72, 129.61, 127.81, 127.55, 113.12, 56.15, 54.99,52.28, 49.58, 49.06, 41.82, 36.17, 34.97, 33.41, 33.09, 31.32, 27.39,23.16, 22.68, 22.39, 20.56, 18.95, 18.54, 11.66, 6.02, 5.0

Solid Support with Immobilized Thiocholesterol (28)

Succinate 27 (2.1 g, 1.9 mmol) was dissolved in dichloroethane (8 mL).To that solution DMAP (0.228 g, 1.9 mmol) was added.2,2′-Dithio-bis(5-nitropyridine) (0.58 g, 1.9 mmol) inacetonitrile/dichloroethane (3:1, 8 mL) was added successively. To theresulting solution triphenylphosphine (0.49 g, 1.9 mmol) in acetonitrile(4 ml) was added. The reaction mixture turned bright orange in color.The solution was agitated briefly using wrist-action shaker (5 mins).Long chain alkyl amine-CPG (LCAA-CPG) (12 g, 1860 μmoles, 155 μm/g) wasadded. The suspension was agitated for 4 h. The CPG was filtered througha sintered funnel and washed with acetonitrile, dichloromethane andether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The loading capacity of the CPG 28 was measured bytaking UV measurement. (57 μM/g).

Synthesis of 4-hydroxy-L-prolinol cholesterol-phosphoramidite (N-alkyllinkage) (33)

[6-(4-Hydroxy-2-hydroxymethyl-pyrrolidin-1-yl)-hexyl]carbamic acidbenzyl ester (29)

Referring to scheme 10, compound 3a (7 g, 19 2 mmol) was dissolved inanhydrous THF and cooled to 0° C. under argon atmosphere. Borane-THF (50mL, 1M soln. in THF, 2.5 equiv.) was added slowly over a period of15mins. The reaction mixture was brought to room temperature and stirredat reflux temperature for over night. After 16 h, the reaction mixturewas cooled and concentrated under vacuum to dryness. To the residue,saturated solution of ammonium chloride (200 mL) was added and theproduct extracted with ethyl acetate (3×100 mL). The combined organiclayer was dried over anhydrous sodium sulfate, filtered and concentratedunder reduced pressure. The crude was purified by column chromatographyover silica gel to afford compound 29 as a viscous liquid (6.2 g, 92%).

¹H NMR (400 MHz, DMSO-d₆): δ 7.33 (m, 5H), 5.1 (s, 2H), 4.94 (d, OH, D₂Oexchangeable, 4.76 (t, OH, D₂O exchangeable) 3.68 (m, 1H), 3.95 (m, 2H),2.92-3.0 (m, 4H), 2.1-2.3 (m, 3H), 1.7-2.0 (2H), 1.34-1.52 (m, 6H),1.2-1.3 (m, 4H).

¹³C NMR (100 MHz, DMSO-d₆): 156.1, 137.3, 128.3, 127.7, 68.2, 65.1,61.9, 57.5, 56.2, 55.1, 36.1, 34.2, 29.3, 26.1, 25.9, 24.6, 24.1.

(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-hexyl)-carbamicacid benzyl ester (30)

Compound 29 (6 g, 17 mmol) was co-evaporated with anhydrous pyridinethree times and then dissolved in pyridine (60 mL). To this solutiondimethylamino pyridine (0.207 g, 1.7 mmol) and DMT-Cl (6 g, 17.9 mmol,1.05 equiv.) were added at room temperature. The reaction mixture wasstirred at room temperature for 16 h. The excess DMT-Cl was quenched bythe addition of methanol (25 mL). The solution was dried under reducedpressure. To the residue was suspended in ethyl acetate (300 mL) andwashed with saturated bicarbonate solution, brine and water. The organiclayer was dried over anhydrous sodium sulfate, filtered and evaporated.24.2 g of the crude product was obtained after removal of the solvent.Upon purification over silica gel using 2% MeOH/DCM compound 30 (8.7 g,79%) was obtained as white foamy solid.

¹H NMR (400 MHz, DMSO-d₆): δ 7.18-7.38 (m, 14H), 6.2-6.5 (m, 4H), 5.0(s, 2H), 4.9 (d, —OH, D₂O exchangeable), 4.4 (m, 1H), 4.15 (m, 1H), 3.7(s, 6H), 3.56 (m, 1H), 3.32 (m, 1H), 3.14 (m, 1H), 2.9-3.0 (m, 6H), 2.18(m, 2H), 1.8-2.1 (m, 2H), 1.1-1.5 (m, 6H).

1-(6-Amino-hexyl)-5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-pyrrolidin-3-ol(31)

Compound 30 (6.52 g, 10 mmol) was dissolved in ethyl acetate (100 mL)and purged with argon. To the solution was added 10% palladium on carbon(2 g). The flask was purged with hydrogen 2 times and stirred further atroom temperature under hydrogen atmosphere for overnight. Thedisappearance of the starting material was confirmed by the TLC. Thereaction mixture was filtered through a pad of Celite and washed withethyl acetate. The combined organic layer was concentrated under reducedpressure to afford compound 31 (4.8 g, 93%) as white solid. This wasused as such for the next step.

(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-hexyl)-carbamicacid10,13-dimethyl-17-octyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester (32)

Compound 31 (4.5 g, 8.67 mmol) was dissolved in anhydrousdichloromethane (100 mL) and cooled to 0° C. To the solution were addedtriethylamine (2.52 g, 3.36 mL, 25 mmol) and cholesteryl chloroformate(3.89 g, 8.67 mmol) successively. The reaction temperature was broughtto ambient temperature and stirred further for 2 h. The completion ofthe reaction was ascertained by TLC (10% MeOH/CHCl₃). The reactionmixture was evaporated under the vacuum to afford the crude product.Compound 32 (3.05 g, 37%) was obtained as a white foamy solid aftercolumn chromatography over silica gel.

¹H NMR (400 MHz, DMSO-d₆): δ 7.1-7.4 (m, 9H), 6.8 (m, 4H), 5.25 (b, 1H),4.65 (s, 1H), 5.35 (bs, 1H), 4.05 (m, 1H), 3.65 (s, 6H), 3.32 9s, 1H),3.14 (m, 2H), 2.6-2.9 (m, 8H), 2-2.2 (m, 4H), 0.6-1.8 (m, 48H).

¹³C NMR (100 MHz, DMSO-d₆): 157.922, 148.38, 140.26, 138.89, 129.78,129.02, 127.74, 127.55, 112.87, 85.41, 67.72, 59.91, 55.1, 54.97, 54.83,22.53, 22.34, 20.87, 19.22, 14.18.

4-hydroxy-L-prolinol-cholesterol-phosphoramidite (N-alkyl linkage) (33)

Compound 32 (2.0 g, 2.14 mmol) was coevaporated with anhydrous toluene(25 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.118 g,1.05 mmol) was added and the mixture was dried over P₂O₅ in a vacuumoven for overnight at 40° C. The reaction mixture was dissolved indichloromethane (5 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.97 g, 1.1 mL,3.22 mmol) was added. The reaction mixture was stirred at ambienttemperature for overnight. The completion of the reaction wasascertained by TLC(R_(f)=0.5 in 1:1 ethyl acetate:hexane). The reactionmixture was diluted with dichloromethane (50 mL) and washed with 5%NaHCO₃ (50 mL) and brine (50 mL). The organic layer was dried overanhydrous Na₂SO₄ filtered and concentrated under reduced pressure. Theresidue was purified over silica gel (50:49:1,EtOAc:Hexane:triethlyamine) to afford 33 as white solid (2.1 g, 86%).

¹H NMR (400 MHz, C₆D₆): δ 7.72 (m, 2H), 7.56 (m, 3H), 7.21 (m, 2H),7-7.1 (m, 3H), 6.8 (m, 3H), 5.4 (bs, 1H), 4.94 (bs, 1H), 4.56 (m, 1H),3.54 (m, 3H), 3.42 (m, 1H), 3.2-3.38 (m, 9H), 3.1 (m, 2H), 2.94 (m, 1H),2.78 (m, 1H), 2.68 (m, 2H), 2.4-2.6 (m, 3H), 2.22 (m, 1H), 2-2.12 (m,8H), 0.9-1.9 (m, 63H), 0.66 (s, 3H).

³¹P NMR (161.82 MHz, C₆D₆): δ 145.48, 145.33 (NO rotamers observed afterremoving amide bond)

¹³C NMR (100 MHz, C₆D₆): δ 159.07, 155.86, 146.20, 140.19, 140.19,137.83, 136.95, 130.65, 129.27, 128.77, 128.51, 127.55, 126.93, 125.64,126.66, 117.5, 113.5, 86.51, 74.63, 72.62, 72.44, 67.37, 63.39, 58.64,58.46, 56.90, 56.46, 54.72, 50.25, 44.84, 44.72, 48.38, 43.41, 43.26,43.29, 42.55, 40.10, 39.0, 39.38, 37.31, 36.8, 36.63, 36.19, 32.27,32.14, 29.41, 28.86, 28.61, 28.38, 27.60, 27.01, 24.74, 24.67, 24.62,24.56, 24.51, 24.32, 24.07, 24.01, 23.00, 22.74, 21.37, 21.33, 20.03,20.0, 19.97, 19.47, 19.01, 12.05.

Synthesis of Solid Support with Immobilized Cholesterol (N-AlkylLinkage) (35)

Succinic acidmono-{5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-[6-(10,13-dimethyl-17-octyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino)-hexyl]pyrrolidin-3-yl}ester(34)

Referring to scheme 11, Compound 33 (1 g, 1.07 mmol) was mixed withsuccinic anhydride (0.16 g, 1.61 mmol) and DMAP (0.012 g, 0.1 mmol) anddried in a vacuum at 40° C. overnight. The mixture was dissolved inanhydrous dichloromethane (10 mL), triethylamine (0.328 g, 0.45 mL, 3.25mmol) was added and the solution was stirred at room temperature underargon atmosphere for 16 h. It was then diluted with dichloromethane (50mL) and washed water (2×25 mL). The organic phase was dried overanhydrous sodium sulfate and concentrated to dryness. The product 34 wasused as such for next step without further purification (1.2 g,Quantitative).

¹H NMR (400 MHz, CDCl₃): δ 7.32-7.36 (m, 2H), 7.2-7.28 (m, 7H), 6.76-6.8(m, 4H), 5.4 (bs, 1H), 4.46 (m, 2H), 3.78 (s, 6H), 3.42 (m, 1H), 3-3.18(m, 3H), 2.5-2.6 (m, 3H), 2.12-2.38 (m, 6H), 1.78-2.02 (m, 7H), 0.8-1.6(m, 42H), 0.66 (s, 3H)

¹³C NMR (100 MHz, CDCl₃): δ 158.78, 158.62, 145.16, 139.8, 136.39,136.22, 130.18, 130.14, 128.23, 128.0, 126.97, 122.91, 113.28, 56.88,56.32, 55.45, 55.4, 50.19, 45.47, 42.51, 39.93, 39.72, 38.67, 37.14,36.74, 36.38, 36.0, 32.1, 32.06, 28.44, 28.22, 24.5, 24.0, 23.04, 22.77,21.24, 19.55, 18.92, 12.07, 8.72

Solid Support with Immobilized Cholesterol (N-Alkyl Linkage) (35)

Succinate 34 (1.2 g, 1.16 mmol) was dissolved in dichloroethane (5 mL).To that solution DMAP (0.142 g, 1.16 mmol) was added.2,2′-Dithio-bis(5-nitropyridine) (0.347 g, 1.16 mmol) inacetonitrile/dichloroethane (3:1, 5 mL) was added successively. To theresulting solution triphenylphosphine (0.304 g, 1.15 mmol) inacetonitrile (2.5 ml) was added. The reaction mixture turned brightorange in color. The solution was agitated briefly using wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (6 g, 900 mmoles,155 μm/g) was added. The suspension was agitated for 4 h. The CPG wasfiltered through a sintered funnel and washed with acetonitrile,dichloromethane and ether successively. Unreacted amino groups weremasked using acetic anhydride/pyridine. The loading capacity of the CPG35 was measured by taking UV measurement. (63 μM/g).

Synthesis of Hydroxy-Prolinol-Phthalimido Phosphoramidite (45)

Compound 37:

Referring to scheme 12, compound 36 (15 g, 60 mmol) was co-evaporatedwith anhydrous pyridine three times and then dissolved in pyridine (200mL). To this solution dimethylamino pyridine (0.733 g, 6 mmol) andDMT-Cl (21.2 g, 62.6 mmol, 1.05 equiv.) were added at room temperature.The reaction mixture was stirred at room temperature for 16 h. Theexcess DMT-Cl was quenched by the addition of methanol (50 mL). Thesolution was dried under reduced pressure. To the residue was suspendedin ethyl acetate (500 mL) and washed with saturated bicarbonatesolution, brine and water. The organic layer was dried over anhydroussodium sulfate, filtered and evaporated. Upon purification over silicagel using 3% MeOH/DCM compound 37 (33 g, 77%) was obtained as whitefoamy solid.

¹H NMR (400 MHz, DMSO-d₆): δ7.22-7.38 (m, 8H), 7.16-7.2 9m, 5H), 7.06(m, 1H), 6.84 (m, 4H), 5.34 (bs, 1H), 4.88-4.96 (m, 2H), 4.25 (m, 1H), 4(bs, 1H), 3.7 (s, 6H), 3.4 (m, 2H), 3.04 (m, 2H), 1.86 (m, 2H).

¹³C NMR (10 MHz, DMSO-d₆): δ 158.0, 154.28, 154.22, 149.62, 145.04,137.15, 136.64, 135.74, 136.67, 129.58, 129.53, 128.4, 128.26, 127.81,127.73, 127.55, 127.29, 126.65, 126.65, 123.91, 113.12, 85.24, 85.14,68.45, 67.83, 65.96, 65.64, 64.39, 63.4, 54.99, 37.67, 36.68.

5-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-pyrrolidin-3-ol (38)

Compound 37 (8.25 g, 14 9 mmol) was dissolved in methanol (20 mL) andpurged with nitrogen. To the solution were added ammonium formate (14.1g, 223 mmol) and 10% Pd/C (0.825 g). The suspension was stirred at roomtemperature for 1 h. The reaction mixture was filtered through a pad ofCelite and washed with methanol. The solution was concentrated todryness under vacuum. The residue was dissolved in ethyl acetate 9250mL) and washed with water (2×25 mL). The organic layer was dried oversodium sulfate, filtered and evaporated to dryness under reducedpressure. Product 38 (6.25 g, 98%) was used without purification for thenext step.

¹H NMR (400 MHz, DMSO-d₆): δ 8.28 (bs, 1H), 7.36 (m, 2H), 7.18-7.3 (m,7H), 6.84 (d, 4H), 4.2 (m, 1H), 3.7 (s, 6H), 3.6 (m, 1H), 3.02 (m, 3H),2.8 (d, 1H), 1.74 (dd, 1H), 1.48 (m, 1H).

¹³C NMR (10 MHz, DMSO-d₆): δ 165.02, 158.06, 149.9, 135.5, 129.74,127.81, 127.72, 126.67, 113.15, 85.58, 69.6, 59.76, 56.81, 55.02,53.6237.66, 14.08.

6-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-hexanoic acid (41a)

6-amino hexanoic acid (39a) (13.1 g, 100 mmol) and phthalic anhydride(40) (14.8 g, 100 mmol) were mixed in toluene (150 mL). To thesuspension was added triethyl amine (13 mL). The suspension was refluxedusing Dean-stark for 16 h. When collection of water ceased, the reactionwas cooled and evaporated to dryness. The residue was suspended in waterand conc. hydrochloric acid (1.5 mL) was added. The suspension wasstirred for 30 mins and filtered. The precipitate was washed with waterand dried over sodium sulfate to afford compound 41a (24.5 g, 93%) whichwas used as such for the next step.

6-(1,3-Dioxo-1,3-dihydro-isoindo1-2-yl)-hexanoic acid pentafluorophenylester (43a)

Referring to scheme 12, compound 41a (13.3 g, 51 mmol) was dissolved inanhydrous dichloromethane (40 mL) and cooled to 0° C. under argon. Tothe solution were added diisopropyl carbodiimide (6.31 g, 7.7 mL, 50mmol) and pentafluoro phenol (42, 9.2 g, 50 mmol). After overnight thereaction mixture was evaporated to dryness. To the residue ethyl acetate(100 mL) was added and the filtered to remove diisopropyl urea. Theprecipitate was washed with ethyl acetate (50 mL). The combined organiclayer was washed with saturated sodium bicarbonate and water. Theorganic layer was dried over sodium sulfate, filtered and evaporated todryness. Compound 43a (R_(f)=0.8 in 10% EtOAc/Hexane, 21.65 g, 92%) wasobtained, which was directly used for the next step without furtherpurification.

¹H NMR (400 MHz, CDCl₃): δ 7.82 (m, 2H), 7.7 (m, 2H), 3.7 (t, 2H), 2.65(t, 2H), 1.7-1.85 (m, 4H), 1.48 (m, 2H).

¹³C NMR (100 MHz, CDCl₃): δ 169.46, 168.62, 142.5, 140.84, 139.29,138.32, 136.75, 134.13, 132.28, 123.39, 37.78, 33.3, 28.33, 26.21,24.46.

2-(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-isoindole-1,3-dione(44a)

Amine 38 (9.32 g, 22.2 mmol) and triethyl amine (4.55 g, 6.27 mL, 45mmol) weres dissolved in anhydrous dichloromethane (20 mL) and cooled to0° C. under argon. To that solution was added compound 43a (9.5 g, 22.2mmol) at 0° C. The reaction mixture was brought to ambient temperatureand stirred further. After 30 mins, disappearance of starting materialswere ascertained by TLC. (10% MeOH/CHCl₃). The reaction mixture wasdiluted with dichloromethane (100 mL) and washed with 5% NaOH solution(3×50 mL) followed by water and brine. The organic layer was dried overanhydrous sodium sulfate, filtered and evaporated to dryness. Uponpurification over silica gel compound 44a was obtained as foamy whitesolid in good yield. (13.2 g, 89%).

¹H NMR (400 MHz, DMSO-d₆): δ 7.84 (m, 4H), 7.3 (m, 4H), 7.18 (m, 5H),6.86 (m, 4H), 4.98 (d, —OH), 4.38 (m, 1H), 4.1 (m, 1H), 3.72 (d, 6H),3.55 (m, 3H), 3.3 (m, 2H), 3.12 (m, 1H), 2.97 (m, 1H), 2.2 (t, 2H), 2.0(m, 1H), 1.9 (m, 1H), 1.82 (m, 1H), 1.44-1.6 (m, 1H), 1.3 (m, 2H), 1.14(m, 1H),

¹³C NMR (100 MHz, DMSO-d₆): δ 172.78, 172.11, 168.74, 168.67, 158.74,158.55, 158.54, 145.24, 144.72, 136.46, 136.27, 137.87, 135.84, 134.18,134.13, 134.09, 132.3, 132.27, 130.18, 130.11, 129.33, 128.22, 128.20,128.08, 128.03, 127.93, 127.12, 126.89, 123.41, 123.38, 113.35, 113.2,86.7, 86.06, 70.7, 69.46, 65.51, 63.67, 56.61, 56.0, 55.9, 55.42, 55.36,54.2, 38.44, 38.0, 37.98, 36.9, 35.0, 33.4, 28.6, 28.5, 28.4, 26.79,26.71, 25.0, 24.6, 24.5.

4-Hydroxy-prolinol-phthalimido phosphoramidite (45a)

Compound 44a (9.0 g, 13.57 mmol) was coevaporated with anhydrous toluene(50 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.766 g,6 8 mmol) was added and the mixture was dried over P₂O₅ in a vacuum ovenfor overnight at 40° C. The reaction mixture was dissolved indichloromethane (20 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (6.13 g, 6.7 mL,20.35 mmol) was added. The reaction mixture was stirred at ambienttemperature for overnight. The completion of the reaction wasascertained by TLC(R_(f)=0.7 in 1:1 ethyl acetate:hexane). The reactionmixture was diluted with dichloromethane (100 mL) and washed with 5%NaHCO₃ (100 mL) and brine (100 mL). The organic layer was dried overanhydrous Na₂SO₄ filtered and concentrated under reduced pressure. Theresidue was purified over silica gel (50:49:1,EtOAc:Hexane:triethlyamine) to afford compound 45a as white solid (10.5g, 89%).

¹H NMR (400 MHz, C₆D₆): δ 7.62 (m, 2H), 7.42 (m, 6H), 7.22 (t, 2H), 7.08(m, 1H), 6.88 (dd, 2H), 6.78 (m, 4H), 4.66 (m, 1H), 4.56 (m, 1H), 3.72(m, 1H), 3.5 (m, 5H), 3.3 (m, 7H), 3.22 (m, 1H), 2.1 (m, 5H), 1.74 (m,4H), 1.56 (m, 2H), 1.26 (m, 2H), 1.1 (m, 13H).

³¹P NMR (161.82 MHz, C₆D₆): δ 145.98, 145.8, 145.63, 146.3 (Rotamersobserved after due to amide bond at the ring)

¹³C NMR (100 MHz, C₆D₆): δ 171.03, 170.08, 167.98, 159.23, 159.0, 146.1,136.76, 136.69, 136.64, 136.27, 133.35, 132.7, 130.59, 130.54, 130.46,128.65, 128.56, 127.55, 126.97, 128.24, 128.0, 127.7, 122.84, 113.62,113.53, 113.51, 86.57, 86.51, 72.67, 72.5, 72.33, 64.48, 58.59, 58.46,58.41, 58.28, 57.77, 56.03, 55.97, 54.81, 54.73, 43.47, 43.35, 37.87,36.42, 36.32, 34.94, 34.88, 33.37, 28.77, 26.94, 24.67, 24.6, 24.51,20.10, 20.04, 19.98.

Compound 45b:

The phosphoramidite 45b is obtained from 39b in four steps as describedfor the synthesis of compound 45a from 39a.

Synthesis of Solid Support Immobilized with Phthalimido Group (46a)

Referring to scheme 13, Compound 44a (3 g, 4.5 mmol) was mixed withsuccinic anhydride (0.675 g, 6.75 mmol) and DMAP (0.055 g, 0.45 mmol)and dried in a vacuum at 40° C. overnight. The mixture was dissolved inanhydrous dichloromethane (10 mL), triethylamine (1.37 g, 1.8 mL, 13 5mmol) was added and the solution was stirred at room temperature underargon atmosphere for 16 h. It was then diluted with dichloromethane (150mL) and washed with 5% ice-cold citric acid (2×50 mL) followed by water(2×50 mL) and brine. The organic phase was dried over anhydrous sodiumsulfate and concentrated to dryness. The succinate was obtained afterpurification over silica gel (3.2 g, 93%).

¹H NMR (400 MHz, DMSO-d₆): δ 8.08 (m, 1H), 7.82 (m, 3H), 7.28 (m, 4H),7.16 (m, 5H), 6.84 (m, 4H), 5.32 (m, 1H), 4.18 (m, 1H), 3.7 (s, 6H),3.53 (m, 3H), 3.32 (m, 2H), 3.2 (m, 1H), 3.0 (m, 1H), 2.94 (s, 2H), 2.4(m, 6H), 2.2 (m, 3H), 2.0 (m, 1H), 1.5 (m, 4H), 1.28 (m, 2H), 1.16 (m,1H).

¹³C NMR (100 MHz, DMSO-d₆): δ 172.95, 171.95, 168.78, 168.64, 158.77,158.59, 145.18, 144.67, 136.44, 136.23, 135.8, 134.13, 133.33, 132.29,130.21, 130.13, 128.25, 128.13, 127.98, 126.94, 123.45, 113.40, 113.26,106.61, 86.11, 73.59, 63.67, 55.76, 55.39, 53.31, 39.64, 38.03, 35.1,35.51, 28.56, 26.82, 24.45.

The succinate (2.7 g, 3.5 mmol) was dissolved in dichloroethane (15 mL).To that solution DMAP (0.0427 g, 3.5 mmol) was added.2,2′-Dithio-bis(5-nitropyridine) (1.086 g, 3.5 mmol) inacetonitrile/dichloroethane (3:1, 15 mL) was added successively. To theresulting solution triphenylphosphine (0.918 g, 3.5 mmol) inacetonitrile (7 ml) was added. The reaction mixture turned bright orangein color. The solution was agitated briefly using wrist-action shaker (5mins). Long chain alkyl amine-CPG (LCAA-CPG) (10.5 g, 1620 mmoles, 155μm/g) was added. The suspension was agitated for 4 h. The CPG wasfiltered through a sintered funnel and washed with acetonitrile,dichloromethane and ether successively. Unreacted amino groups weremasked using acetic anhydride/pyridine. The loading capacity of the CPG46a was measured by taking UV measurement. (63 μM/g).

Synthesis of Solid Support Immobilized with Phthalimido Group (46b)

The desired compound 46b is obtained from compound 44b in two steps asdescribed for the preparation of compound 46a from the correspondingprecursor 44a.

Serinol as a Linker:

Synthesis of Solid Support Immobilized with Cholesterol—Serinol Linker(54)

ε-N-cholesteryloxycarbonylaminocaproic acid (49)

Referring to Scheme 14, c-aminocaproic acid (3.93 g, 30 mmol) wassuspended in pyridine (60 mL). The flask was flushed with nitrogen andto the mixture was added N,O-bis(trimethylsilyl)acetamide (10 mL, 70mmol) under stirring. The reaction mixture was stirred at roomtemperature for 30 min. Then cooled in ice bath. Cholesterylchloroformate (13.5 g, 30 mmol) was added into reaction mixture in twoportions over 2 h. The reaction was continued by stirring at roomtemperature for another 4 h. 2% HCl aqueous solution (150 ml) was addedunder cooling with ice bath. The mixture was stirred for 5 min. and thenpoured into a separating funnel. The product was extracted withdichloromethane (3×150 mL) The combined organic layer was washed with 2%HCl solution (2×150 mL) and with brine (2×150 mL), dried over anhydroussodium sulfate, filtered and evaporated to dryness giving a yellow foam(14.44 g, 87%)

¹H NMR (400 MHz, CDCl₃): δ 5.36 (m, 1H), 4.48 (m, 1H), 3.15 (m, 2H),2.38 (t, 2H), 1.8-2.04 (m, 5H), 1.32-1.7 (m, 19H), 0.88-1.2 (m, 22H),0.67 (s, 3H)

¹³C NMR (100 MHz, CDCl₃): δ 179.17, 156.42, 139.94, 126.64, 74.43,56.82, 56.28, 50.14, 42.45, 39.88, 39.67, 38.69, 37.13, 36.69, 36.34,35.97, 34.08, 32.05, 32.01, 29.78, 28.48, 28.30, 28.17, 26.32, 24.47,24.44, 24.0, 23.0, 22.73, 21.19, 19.5, 18.87, 12.01.

ε-N-Pentalfluorophenyl Cholesteryloxycarbonylamino Caproate (50)

Referring to scheme 14, ε-N-cholesteryloxycarbonylaminocaproic acid (49)(22.71 g, 41.9 mmol) was dissolved in anhydrous dichloromethane (40 mL)and cooled to 0° C. To the solution were added diisopropyl carbodiimide(5.17 g, 6.4 mL, 41 mmol) and triethylamine (10.2 g, 13.7 mL, 100 mmol).After stirring for 20 mins at 0° C., pentafluorophenol (7.71 g, 41.9mmol) was added and the stirring was continued at room temperature underargon for over night. The reaction mixture was evaporated to dryness. Tothe residue ethyl acetate (100 mL) was added and the filtered to removediisopropyl urea. The precipitate was washed with ethyl acetate (50 mL).The combined organic layer was washed with saturated sodium bicarbonateand water. The organic layer was dried over sodium sulfate, filtered andevaporated to dryness. Compound 50 (R_(f)=0.7 in 10% EtOAc/hexane, 25.4g, 86%) was obtained, which was directly used for the next step withoutfurther purification.

¹H NMR (400 MHz, CDCl₃): δ 5.38 (m, 1H), 4.47 (m, 1H), 3.2 (m, 2H), 2.36(t, 2H), 1.81-2.05 (m, 5H), 1.3-1.7 (m, 19H), 0.89-1.21 (m, 22H), 0.68(s, 3H)

¹³C NMR (100 MHz, CDCl₃): δ 179.2, 156.2, 139.84, 139.29, 138.32,136.75, 134.13, 132.28, 126.64, 123.39, 74.43, 56.82, 56.28, 50.14,42.45, 39.88, 39.67, 38.69, 37.13, 36.69, 36.34, 35.97, 34.08, 32.05,32.01, 29.78, 28.48, 28.30, 28.17, 26.32, 24.47, 24.44, 24.0, 23.0,22.73, 21.19, 19.5, 18.87, 12.01.

Synthesis of[5-(2-Hydroxy-1-hydroxymethyl-ethylcarbamoyl)-pentyl]carbamic acid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester (52)

Serinol (51) (1.37 g, 15 mmol) and triethyl amine (3.03 g, 4.15 mL, 30mmol) were dissolved in anhydrous dichloromethane (20 mL) and cooled to0° C. under argon. To that solution was added compound 50 (7.1 g, 10mmol) at 0° C. The reaction mixture was brought to ambient temperatureand stirred further. After 3 h, disappearance of starting materials wereascertained by TLC. (10% MeOH/CHCl₃). The reaction mixture was dilutedwith dichloromethane (100 mL) and washed with 5% NaOH solution (3×50 mL)followed by water and brine. The organic layer was dried over anhydroussodium sulfate, filtered and evaporated to dryness. Upon purificationover silica gel using 5% MeOH/DCM, compound 52 was obtained as foamywhite solid in good yield. (5.61 g, 90%).

¹H NMR (400 MHz, DMSO-d₆): δ 5.32 (m, 1H), 4.58 (t, 2H), 4.28 (m, 2H),3.58 (m, 1H), 3.38 (m, 4H), 2.91 (t, 2H), 2.2 (m, 2H), 2.06 (t, 2H),1.72-1.98 (m, 5H), 0.82-1.58 (m, 37H), 0.74 (s, 3H).

M/S(m/z): Calculated: 616.48 0 bserved: 617.5 (M⁺+1), 636.4 (M⁺+Na).

Synthesis of(5-{1-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-2-hydroxy-ethylcarbamoyl}-pentyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester (54)

Diol 52 (5.6 g, 9.1 mmol) was co-evaporated with anhydrous pyridinethree times and then dissolved in pyridine (10 mL). To this solutiondimethylamino pyridine (0.110 g, 0.91 mmol) and DMT-Cl (3.23 g, 9.53mmol, 1.05 equiv.) were added at room temperature. The reaction mixturewas stirred at room temperature for 16 h. Due to the presence of twoprimary hydroxyl groups, the reaction never went to completion. Thesolution was dried under reduced pressure and co-evaporated with tolueneto remove residual pyridine. To the residue was suspended in ethylacetate (200 mL) and washed with saturated bicarbonate solution, brineand water. The organic layer was dried over anhydrous sodium sulfate,filtered and evaporated. The crude product was obtained after removal ofthe solvent. Upon purification over silica gel using 2% MeOH/DCMcompound 53 (0.680 g, 10%) was obtained as white foamy solid.

¹H NMR (400 MHz, DMSO-d₆): δ 7.62 (d, 1H), 7.36 (m, 2H), 7.18-7.3 (m,6H), 7.1 (m, 1H), 6.86 (m, 4H), 5.32 (bs, 1H), 4.6 (t, 1H), 4.28 (m,1H), 3.98 (m, 1H), 3.72 (s, 6H), 3.42 (m, 2H), 2.98 (m, 1H), 2.9 (m,3H), 1.72-2.3 (m, 9H), 0.8-1.58 (m, 39H), 0.64 (s, 3H).

Synthesis of Succinic acidmono-(3-[bis-(4-methoxy-phenyl)-phenyl-methoxy]-2-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoylamino}-propyl)ester (54)

DMT-alcohol 53 (0.650 g, 0.707 mmol) was mixed with succinic anhydride(0.100 g, 1 mmol) and DMAP (0.0123 g, 0.1 mmol) and dried in a vacuum at40° C. overnight. The mixture was dissolved in anhydrous dichloromethane(5 mL), triethylamine (0.203 g, 0.27 mL, 2 mmol) was added and thesolution was stirred at room temperature under argon atmosphere for 16h. It was then diluted with dichloromethane (20 mL) and washed with icecold aqueous citric acid (5% wt., 25 mL) and water (2×25 mL). Theorganic phase was dried over anhydrous sodium sulfate and concentratedto dryness. The crude product was purified by column chromatography toafford compound 54 as white solid (0.54 g, 78% yield; R_(f)=0.5 in 10%MeOH/CHCl₃).

¹H NMR (400 MHz, CDCl₃): δ 7.26-7.32 (m, 5H), 7.16-7.18 (m, 4H), 6.84(m, 4H), 5.38 (bs, 1H), 4.6 (t, 1H), 4.2-4.6 (m, 4H), 3.8 (s, 6H), 3.62(m, 2H), 3.18 (m, 4H), 2.6-2.72 (m, 4H), 2.2-2.38 (m, 3H), 1.82-2.04 (m,9H), 0.84-1.62 (m, 39H), 0.66 (s, 3H).

Synthesis of Cholesterol Immolized on Solid Support with Serinol Linker(55)

Succinate 54 (0.51 g, 0.5 mmol) was dissolved in dichloroethane (2 mL).To that solution DMAP (0.061 g, 0.5 mmol) was added.2,2′-Dithio-bis(5-nitropyridine) (0.155 g, 0 5 mmol) inacetonitrile/dichloroethane (3:1, 2 mL) was added successively. To theresulting solution triphenylphosphine (0.131 g, 0.5 mmol) inacetonitrile (1 ml) was added. The reaction mixture turned bright orangein color. The solution was agitated briefly using wrist-action shaker (5mins). Long chain alkyl amine-CPG (LCAA-CPG) (2.2 g, 115 μm/g) wasadded. The suspension was agitated for 3 h. The CPG was filtered througha sintered funnel and washed with acetonitrile, dichloromethane andether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The loading capacity of the CPG was measured bytaking UV measurement. (35 μM/g).

Serinol-Cholesterol-Phosphoramidite (56)

Referring to Scheme 15, Compound 53 (0.92 g, 1 mmol) is coevaporatedwith anhydrous toluene (25 mL). To the residueN,N-tetraisopropylammonium tetrazolide (0.056 g, 0 5 mmol) is added andthe mixture is dried over P₂O₅ in a vacuum oven for overnight at 40° C.The reaction mixture is dissolved in dichloromethane (25 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.9 g, 2.1 mL,6.3 mmol) is added. The reaction mixture is stirred at ambienttemperature for overnight. The completion of the reaction is ascertainedby TLC. The reaction mixture is diluted with dichloromethane (25 mL) andwashed with 5% NaHCO₃ (50 mL) and brine (50 mL). The organic layer isdried over anhydrous Na₂SO₄ filtered and concentrated under reducedpressure. The residue is purified over silica gel (50:49:1,EtOAc:Hexane:triethlyamine) to afford amidite 56.

Synthesis of Pyrrolidine-Cholesterol Phosphoramidite

Synthesis of 3-(Ethoxycarbonylmethyl-Amino)-Propionic Acid Ethyl Ester(58)

Referring to scheme 16, a 4.7M aqueous solution of sodium hydroxide (50mL) was added into a stirred, ice-cooled solution of ethyl glycinatehydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethylacrylate (23.1 g, 0.23 mole) was added and the mixture was stirred atroom temperature until the completion of reaction was ascertained by TLC(19 h). After 19 h which it was partitioned with dichloromethane (3×100mL). The organic layer was dried with anhydrous sodium sulfate, filteredand evaporated. The residue was distilled to afford 58 (28.8 g, 61%).

¹H NMR (CDCl₃, 400 MHz): δ 4.1-4.2 (m, 4H), 3.4 (s, 2H), 2.8 (t, J=6.7Hz, 2H), 2.4 (t, J=6.7 Hz, 2H), 1.25 (m, 6H).

Synthesis of3-[(6-Benzyloxycarbonylamino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionicacid ethyl ester (59)

6-benzyloxyamino hexanoic acid (13.25 g, 50 mmol) was dissolved inanhydrous dichloromethane (50 mL) and cooled to 0° C. To the solutionwere added diisopropyl carbodiimide (6.31 g, 7.7 mL, 50 mmol) andtriethylamine (10.2 g, 13.7 mL, 100 mmol). After stirring for 20 mins at0° C., compound 58 (10.16 g, 50 mmol) was added and the stirring wascontinued at room temperature under argon for over night. The reactionmixture was evaporated to dryness. To the residue ethyl acetate (100 mL)was added and the filtered to remove diisopropyl urea. The precipitatewas washed with ethyl acetate (50 mL). The combined organic layer waswashed with 2N HCl, saturated sodium bicarbonate and water. The organiclayer was dried over sodium sulfate, filtered and evaporated to dryness.Compound 59 (R_(f)=0.5 in 25% EtOAc/Hexane, 20.5 g) was obtained, whichwas directly used for the next step without further purification.

¹H NMR (CDCl₃, 400 MHz): δ 7.36 (m, 5H), 5.1 (s, 2H), 4.06-4.22 (m, 6H),3.6-3.7 (m, 2H), 3.2 (m, 2H), 2.6 (m, 2H), 2.42 (m, 2H), 2.14 (m, 2H),1.2-1.7 (m, 12H).

Synthesis of1-(6-Benzyloxycarbonylamino-hexanoyl)-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester OD

To a suspension of potassium t-butoxide (7.12 g, 64 mmol) in toluene(150 mL) at 0° C. under nitrogen, was added diester 59 (20 g, 44 mmol)in toluene (25 mL) over a 10 min period. The solution was stirred for 30min at 0° C. and 5 mL of glacial acetic acid was added, immediatelyfollowed by 25 g of NaH₂PO₄.H₂O in 250 mL of ice-cold water. Theresultant mixture was extracted with chloroform (3×200 mL), and thecombined organic extracts were washed twice with phosphate buffer (2×25mL, pH=7.0), dried over anhydrous sodium sulfate and evaporated todryness. The residue was dissolved in toluene (300 mL), cooled to 0° C.,and extracted with cold pH 9.5 carbonate buffer (3×150 mL). The aqueousextracts were converted to pH 3 with phosphoric acid, and extracted withchloroform (5×125 mL) which were combined, dried, and evaporated to aafford keto ester 60 (12 g, 45%).

The toluene fraction was washed with water (25 mL), dried and evaporatedto afford ketoester 61 (7.6 g, 28%).

¹H NMR (CDCl₃, 400 MHz): δ 7.35 (m, 5H), 5.1 (s, 2H), 4.05-4.34 (m, 6H),3.8 (m, 1H), 3.2 (m, 4H), 2.6 (m, 1H), 2.2-2.4 (m, 1H), 1.68 (m, 1H),1.52 (m, 1H), 1.24-1.4 (m, 6H).

Synthesis of1-(6-Benzyloxycarbonylamino-hexanoyl)-4-hydroxy-pyrrolidine-3-carboxylicacid ethyl ester (62)

To a solution of sucrose (3 g) in distilled water (40 mL) was addedBaker's yeast (2 g). The suspension was heated at 32° C. for 1 h. Thecontent of the flask was then poured into a flask containing ketoester60 (4 g, 9.88 mmol, dissolved in 4 mL of methanol). Stirring wascontinued at 32° C. for 24 h after which additional sucrose (3 g) inwarm (40° C.) distilled water was added. After 48 h, the suspension wasfiltered through a pad of Celite. The pad was washed with water and theaqueous layer was extracted with ethyl acetate (3×250 mL). The combinedorganic layer was dried over anhydrous sodium sulfate, filtered andconcentrated under reduced pressure. The residue was subjected to flashchromatography (30% EtOAc/Hexane) to afford alcohol 62 (1.7 g, 42%).

¹H NMR (CDCl₃, 400 MHz): δ 7.32 (m, 5H), 5.12 (s, 2H), 4.56 (m, 1H), 4.2(m, 2H), 3.9 (m, 1H), 3.83 (m, 1H), 3.63 (m, 1H), 3.48 (m, 1H),2.82-3.06 (m, 3H), 2.2 (t, 2H), 1.22-1.41 (m, 9H). (Also observed minorrotamer due to amide bond)

¹³C NMR (CDCl₃, 100 MHz): δ 173.2, 172.5, 171.41, 156.78, 136.74,128.60, 128.17, 128.11, 70.19, 68.38, 66.56, 60.56, 58.22, 57.71, 55.36,54.60, 52.36, 40.78, 37.73, 34.2, 29.64, 24.22, 21.66, 14.29.

Synthesis of[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid benzyl ester (63)

To the solution of lithium borohydride (0.305 g, 13 mmol) in anhydroustetrahydrofuran (25 mL) was added a solution of ethyl ester 62 (3.74 g,9 2 mmol) in THF (25 mL) over a period of 30 mins at 0° C. After theaddition the reaction mixture was brought to room temperature andstirred further under argon. The completion of the reaction wasascertained by TLC after 4 h. (R_(f)=0.4 in 10% MeOH/CHCl₃). Thereaction mixture was evaporated to dryness and cooled to 0° C. To theresidue 3N HCl (40 mL) was added slowly. After stirring for 30 mins theproduct was extracted with dichloromethane (3×75 mL). The combinedorganic layer was washed with brine and dried over sodium sulfate.Organic layer was filtered and evaporated to dryness. Compound 63 waspurified by column chromatography first by eluting withdichloromethane/methanol (5%) (3.2 g, 92%).

¹H NMR (CDCl₃, 400 MHz): δ 7.34 (m, 5H), 5.16 (s, 2H), 4.64 (m, 1H), 4.4(bs, 1H), 4.2 (m, 1H), 3.78 (m, 2H), 3.62 (m, 3H), 3.5 (m, 2H), 2.06 (m,4H), 1.55 (m, 4H), 1.2 (m, 2H).

Synthesis of(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid benzyl ester (64)

Referring to scheme 16, compound 63 (3.65 g, 10 mmol) was co-evaporatedwith anhydrous pyridine three times and then dissolved in pyridine (10mL). To this solution dimethylamino pyridine (0.122 g, 1 mmol) andDMT-Cl (3.55 g, 10.5 mmol, 1.05 equiv.) were added at room temperature.The reaction mixture was stirred at room temperature for 16 h. Theexcess DMT-Cl was quenched by the addition of methanol (10 mL). Thesolution was dried under reduced pressure. To the residue was suspendedin ethyl acetate (200 mL) and washed with saturated bicarbonatesolution, brine and water. The organic layer was dried over anhydroussodium sulfate, filtered and evaporated. The crude product was obtainedafter removal of the solvent. Upon purification over silica gel using 3%MeOH/DCM compound 64 (5.9 g, 88%) was obtained as white foamy solid.

¹H NMR (CDCl₃, 400 MHz): δ 7.24-7.38 (m, 13H), 7.18 (m, 2H), 6.84 (m,3H), 5.1 (s, 2H), 4.96 (m, 1H), 4.36 (m, 2H), 3.74-3.8 (m, 8H), 3.52 (m,2H), 3.2 (m, 3H), 1.88-2.38 (m, 4H), 1.28-1.72 (m, 6H)

¹³C NMR (100 MHz, CDCl₃): δ 174.7, 172.7, 171.9, 171.3, 171.2, 158.8,158.7, 158.6, 158.5, 158.4, 158.3, 156.7, 156.7, 156.6, 147.5, 145.8,145.2, 144.9, 144.7, 144.4, 139.6, 137.1, 137.04, 137.01, 136.9, 136.82,136.78, 136.55, 136.47, 136.45, 136.3, 136.28, 135.93, 135.85, 135.81,130.2, 130.1, 130.0, 129.9, 129.3, 128.69, 128.66, 128.22, 128.16,128.0, 127.99, 127.94, 127.91, 127.77, 113.52, 113.43, 113.35, 113.3,113.24, 113.19, 113.03, 86.8, 86.1, 85.9, 73.0, 71.6, 71.5, 70.5, 69.3,67.3, 67.1, 68.76, 68.71, 64.38, 63.7, 60.58, 60.0, 56.4, 55.8, 55.7,55.45, 55.41, 55.35, 55.33, 40.97, 40.87, 40.77, 37.13, 36.83, 35.13,35.00, 34.81, 34.6, 33.3, 29.8, 26.73, 25.5, 26.4, 26.2, 24.9, 24.6,24.5, 24.3, 24.2, 21.1, 14.3.

Synthesis of6-Amino-1-{3-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-hexan-1-one(65)

Compound 64 (5.9 g, 8.84 mmol) was dissolved in methanol (10 mL) andpurged with argon. To the solution was added 10% palladium on carbon(0.6 g). The flask was purged with hydrogen 2 times and stirred furtherat room temperature under hydrogen atmosphere for overnight. Thedisappearance of the starting material was confirmed by the TLC. Thereaction mixture was filtered through a pad of Celite and washed withmethanol. The combined organic layer was concentrated under reducedpressure to afford compound 65 (4.3 g, 92%) as white solid. This wasused as such for the next step.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.16-7.36 (m, 9H), 6.88 (m, 4H), 4.4 (m,1H), 4.16 (m, 1H), 3.72 (m, 6H), 3.56 (dd, 1H), 3.34 (m, 1H), 3.14 (m,1H), 3.0 (m, 1H), 2.7 (m, 2H), 2.2 (m, 2H), 1.8-2.1 (m, 3H), 1.28-1.58(m, 6H), 1.16 (m, 2H).

¹³C NMR (DMSO-d₆, 100 MHz): δ 170.84 (Minor disappears at 80° C.),170.75, 165.82, 158.1, 157.98, 145.1, 144.76, 135.86, 135.74, 129.61,129.57, 127.91, 127.81, 127.57, 126.61, 113.23, 113.31, 85.79, 85.11,68.55 63.33, 56.76, 55.07, 55.02, 38.63, 36.27, 33.89, 32.34, 27.12,27.05, 23.91, 20.77, 14.09.

Synthesis of(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-4H-cyclopenta[a]phenanthren-3-ylester (66)

Referring to scheme 16, compound 65 (7.75 g, 14 5 mmol) was dissolved inanhydrous dichloromethane (50 mL) and cooled to 0° C. To the solutionwere added triethylamine (3 g, 4.2 mL, 30 mmol) and cholesterylchloroformate (6.5 g, 29 mmol) successively. The reaction temperaturewas brought to ambient temperature and stirred further for 2 h. Thecompletion of the reaction was ascertained by TLC (10% MeOH/CHCl₃). Thereaction mixture was evaporated under the vacuum to afford the crudeproduct. Compound 66 (12.4 g, 88%) was obtained as a white foamy solidafter column chromatography over silica gel using 3% MeOH/DCM.

¹H NMR (400 MHz, DMSO-d₆): δ 7.12-7.3 (m, 8H), 6.95 (m, 1H), 6.84 (m,4H), 5.3 (bs, 1H), 4.92 and 4.84 (d, OH, exchangeable with D₂O),4.21-4.38 (m, 2H), 4.35 (m, 1H), 3.7 (s, 6H), 3.54 (m, 1H), 3.28 (m,2H), 3.12 (m, 1H), 2.84-2.98 (m, 3H), 2.12-2.28 (m, 3H), 1.7-2.0 (m,7H), 0.8-1.52 (m, 40H), 0.6 (s, 3H).

¹³C NMR (100 MHz, DMSO-d₆): δ 170.8, 158.0, 157.9, 155.6, 145.0, 139.7,135.8, 135.7, 129.5, 127.7, 127.5, 121.7, 113.1, 113.0, 85.7, 85.1,72.7, 68.5, 63.3, 56.1, 55.5, 54.9, 49.4, 41.8, 36.5, 35.2, 31.3, 27.7,27.3, 26.0, 24.1, 23.8, 23.2, 22.6, 22.3, 20.5, 18.9, 18.5, 11.6.

M/S (ESI): Calculated: 944.63 0 bserved: 967.6 (M⁺+Na).

Synthesis of Pyrrolidine-Cholesterol Phosphoramidite (67)

Compound 66 (0.15 g, 0.158 mmol) was coevaporated with toluene (5 mL).To the residue N,N-tetraisopropylammonium tetrazolide (0.0089 g, 0.079mmol) was added and the mixture was dried over P₂O₅ in a vacuum oven forovernight at 40° C. The reaction mixture was dissolved in the mixture ofanhydrous acetonitrile/dichloromethane (2;1, 1 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.0714 g,0.0781 mL, 0.237 mmol) was added. The reaction mixture was stirred atambient temperature for overnight. The completion of the reaction wasascertained by TLC (1;1 ethyl acetate:hexane). The solvent was removedunder reduced pressure and the residue was dissolved in ethyl acetate(10 mL) and washed with 5% NaHCO₃ (4 mL) and brine (4 mL). The ethylacetate layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The resulting mixture was chromatographed (50:49:1,EtOAc:Hexane:triethlyamine) to afford 67 as white foam (0.152 g, 84%).

¹H NMR (CDCl₃, 400 MHz): δ 7.36 (m, 2H), 7.24 (m, 7H), 6.8 (m, 4H), 5.38(m, 1H), 4.7 (m, 1H), 4.5 (m, 1H), 4.36 (m, 1H), 3.5-3.8 (m, 9H),3.36-3.6 (m, 4H), 3.14 (m, 3H), 2.58 (m, 2H), 1.8-2.38 (m, 12H),0.84-1.68 (m, 51H), 0.66 (s, 3H).

³¹P NMR (161.82 MHz, CDCl₃): δ 146.3, 146.2, 145.98, 145.8, 145.63,145.4 (multiple peaks due mixer of diastereomer and Rotamers observedafter due to amide bond at the ring)

¹³C NMR (CDCl₃, 100 MHz): δ 171.6, 158.75, 158.58, 156.36, 145.32,144.78, 140.10, 136.48, 136.36, 136.32, 135.84, 130.19, 129.24, 128.44,128.27, 128.21, 128.13, 127.97, 127.15, 126.92, 125.51, 122.62, 117.87,117.79, 113.40, 113.25, 86.16, 86.11, 74.31, 72.39, 63.92, 58.5, 58.3,58.1, 56.8, 56.3, 55.9, 55.8, 55.4, 55.3, 52.2, 43.4, 43.3, 42.5, 40.8,39.9, 39.7, 38.7, 37.2, 36.7, 36.3, 36.0, 35.0, 32.1, 32.0, 30.0, 28.45,28.4, 28.2, 26.8, 24.8, 24.7, 24.69, 24.6, 24.5, 24.0, 23.0, 22.7, 21.6,21.2, 20.6, 20.59, 20.52, 19.5, 18.9, 11.6

Synthesis of Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-4H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)ester (68)

Referring to scheme 17, Compound 66(12 g, 12.69 mmol) was mixed withsuccinic anhydride (1.9 g, 19 mmol) and DMAP (1.56 g, 13 mmol) and driedin a vacuum at 40° C. overnight. The mixture was dissolved in anhydrousdichloromethane (50 mL), triethylamine (2 g, 3.6 mL, 26 mmol) was addedand the solution was stirred at room temperature under argon atmospherefor 16 h. It was then diluted with dichloromethane (100 mL) and washedwith ice cold aqueous citric acid (5% wt., 100 mL) and water (2×100 mL).The organic phase was dried over anhydrous sodium sulfate andconcentrated to dryness. The crude product was purified by columnchromatography to afford compound 68 as white solid (12.1 g, 91% yield;R_(f)=0.5 in 10% MeOH/CHCl₃).

¹H NMR (400 MHz, DMSO-d₆): δ 7.12-7.32 (m, 9H), 6.82 (m, 4H), 5.3 (m,2H), 4.26 (m, 1H), 4.06 (m, 1H), 3.6-3.78 (m, 8H), 3.52 (m, 1H), 3.2 (m,1H), 3 (m, 2H), 2.88 (m, 2H), 2.7 (m, 1H), 2.1-2.24 (m, 6H), 1.84-2.04(m, 3H), 1.75 (m, 4H), 0.8-1.52 (m, 39H), 0.62 (m, 3H)

¹³C NMR (100 MHz, DMSO-d₆): δ 173.42, 171.97, 170.66, 158.12, 157.99,156.63, 144.97, 144.67, 139.77, 135.73, 135.59, 135.38, 129.61, 127.88,127.8, 127.57, 126.61, 121.8, 113.2. 113.12, 85.96, 85.26, 72.81, 72.73,63.24, 56.12, 55.58, 55.0, 54.97, 54.84, 49.47, 41.85, 36.60, 35.66,35.22, 33.09, 31.38, 29.33, 28.84, 28.74, 27.9, 27.8, 27.4, 25.96,24.40, 23.86, 23.23, 22.66, 22.39, 20.57, 18.98, 18.53, 11.66, 10.01.

Synthesis of Cholesterol Immobilized Solid Support with PyrrolidineLinker (69) Succinate 68 (8.4 g, 8 mmol) was dissolved in dichloroethane(40 mL). To that solution DMAP (0.977 g, 8 mmol) was added.2,2′-Dithio-bis(5-nitropyridine) (2.49 g, 8 mmol) inacetonitrile/dichloroethane (3:1, 40 mL) was added successively. To theresulting solution triphenylphosphine (2.1 g, 8 mmol) in acetonitrile(20 ml) was added. The reaction mixture turned bright orange in color.The solution was agitated briefly using wrist-action shaker (5 mins).Long chain alkyl amine-CPG (LCAA-CPG) (30 g, 155 μm/g) was added. Thesuspension was agitated for 4 h. The CPG was filtered through a sinteredfunnel and washed with acetonitrile, dichloromethane and ethersuccessively. Unreacted amino groups were masked using aceticanhydride/pyridine. The loading capacity of the CPG was measured bytaking UV measurement. (82 μM/g).

Synthesis of Phthalimido-Pyrrolidine Phosphoramidite

Synthesis of 3-(Benzyloxycarbonyl-ethoxycarbonylmethyl-amino)-propionicacid ethyl ester (70)

To a solution of diester 58 (3.88 g, 20 2 mmol) in dry acetonitrile (40mL) at 0° C., under Argon, was added slowly benzyl chloroformate (3.17mL, 1.1 equiv.). The solution was stirred at 0° C. for 1 h after whichit was poured into water (50 mL). The phases were separated and theaqueous layer was extracted with dichloromethane (3×50 mL). the combinedorganic extracts were washed with 5% HCl, water, brine and dried overanhydrous sodium sulfate. Evaporation of the solvents was followed bydistillation to afford compound 70 as a colorless oil (5.4 g, 80%).

¹H NMR (400 MHz, CDCl₃): δ 7.18-7.4 (m, 5H), 5.13 (s, 2H), 4.12 (q, 2H,J=7.1 Hz), 4.06 (q, 2H, J=7.1 Hz), 3.58 (t, 2H, J=6.4 Hz), 2.6 (t, 2H,J=6.4 Hz), 1.18 (t, 3H, J=7.1 Hz), 1.2 (t, 3H, J=7.1 Hz).

¹³C NMR (100 MHz, CDCl₃): δ 172.7, 170.4, 156.7, 136.8, 129, 128.9,128.4, 128.3, 128.2, 68, 61.6, 61.1, 50.9, 45.8, 34.6, 14.6.

Synthesis of 4-Oxo-pyrrolidine-1,3-dicarboxylic acid 1-benzyl ester3-ethyl ester (71)

To a suspension of potassium t-butoxide (2.52 g, 22.4 mmol, 1.4 equiv.)in toluene (50 mL) at 0° C. under nitrogen, was added diester 70 (5.41g, 16 mmol) in toluene (10 mL) over a 10 min period. The solution wasstirred for 30 min at 0° C. and 2 mL of glacial acetic acid was added,immediately followed by 10 g of NaH₂PO₄.H₂O in 100 mL of ice-cold water.The resultant mixture was extracted with chloroform (3×150 mL), and thecombined organic extracts were washed twice with phosphate buffer (2×25mL, pH=7.0), dried over anhydrous sodium sulfate and evaporated todryness. The residue was dissolved in toluene (200 mL), cooled to 0° C.,and extracted with cold pH 9.5 carbonate buffer (3×150 mL). The aqueousextracts were converted to pH 3 with phosphoric acid, and extracted withchloroform (5×125 mL) which were combined, dried, and evaporated to aafford keto ester 71 (2.2 g, 42%).

The toluene fraction was washed with water (10 mL), dried and evaporatedto afford ketoester 72 (1.3 g, 24%).

¹H NMR (400 MHz, CDCl₃): δ 7.2 (m, 5H), 5.18 (s, 2H), 4.25 (m, 4H), 4.1(m, 1H), 3.94 (m, 1H), 3.62 (m, 1H), 1.3 (m, 3H).

¹³C NMR (100 MHz, CDCl₃): δ 203.29, 166.14, 153.56, 136.36, 127.89,127.34, 127.04, 65.96, 60.75, 52.77, 51.84, 45.55, 13.41

Synthesis of 4-Hydroxy-pyrrolidine-1,3-dicarboxylic acid 1-benzyl ester3-ethyl ester (73)

To a solution of sucrose (3 g) in distilled water (40 mL) was addedBaker's yeast (2 g). The suspension was heated at 32° C. for 1 h. Thecontent of the flask was then poured into a flask containing ketoester71 (2.9 g, 9.88 mmol, dissolved in 4 mL of methanol). Stirring wascontinued at 32° C. for 24 h after which additional sucrose (3 g) inwarm (40° C.) distilled water was added. After 48 h, the suspension wasfiltered through a pad of Celite. The pad was washed with water and theaqueous layer was extracted with ethyl acetate (3×250 mL). The combinedorganic layer was dried over anhydrous sodium sulfate, filtered andconcentrated under reduced pressure. The residue was subjected to flashchromatography (30% EtOAc/Hexane) to afford alcohol 73 (1.2 g, 41%).

¹H NMR (CDCl₃, 400 MHz): δ 7.36 (m, 5H), 5.1 (m, 3H), 4.1 (m, 3H), 3.88(m 2H), 3.5 (m, 1H), 3.34 (m, 2H), 1.2 (m, 3H) (Also observed minorrotamer due to amide bond).

Synthesis of 3-Hydroxy-4-hydroxymethyl-pyrrolidine-1-carboxylic acidbenzyl ester (74)

To the solution of lithium borohydride (0.305 g, 13 mmol) in anhydroustetrahydrofuran (25 mL) was added a solution of ethyl ester 73 (2.69 g,9 2 mmol) in THF (25 mL) over a period of 30 mins at 0° C. After theaddition the reaction mixture was brought to room temperature andstirred further under argon. The completion of the reaction wasascertained by TLC after 4 h. (R_(f)=0.3 in 10% MeOH/CHCl₃). Thereaction mixture was evaporated to dryness and cooled to 0° C. To theresidue 3N HCl (40 mL) was added slowly. After stirring for 30 mins theproduct was extracted with dichloromethane (3×75 mL). The combinedorganic layer was washed with brine and dried over sodium sulfate.Organic layer was filtered and evaporated to dryness. Compound 74 waspurified by column chromatography first by eluting withdichloromethane/methanol (5%) (1.98 g, 85%).

¹H NMR (CDCl₃, 400 MHz): δ 7.36 (m, 5H), 5.16 (s, 2H), 4.62 (m, 1H), 4.4(bs, 1H), 4.2 (m, 1H), 3.8 (m, 2H), 3.64 (m, 3H), 3.5 (m, 2H). (Alsoobserved minor rotamer due to amide bond).

Synthesis of3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidine-1-carboxylicacid benzyl ester (75)

Referring to scheme 18, compound 74 (4.39 g, 17.5 mmol) wasco-evaporated with anhydrous pyridine three times and then dissolved inpyridine (30 mL). To this solution dimethylamino pyridine (0.213 g, 1.75mmol) and DMT-Cl (6.22 g, 18.4 mmol, 1.05 equiv.) were added at roomtemperature. The reaction mixture was stirred at room temperature for 16h. The excess DMT-Cl was quenched by the addition of methanol (10 mL).The solution was dried under reduced pressure. To the residue wassuspended in ethyl acetate (300 mL) and washed with saturatedbicarbonate solution, brine and water. The organic layer was dried overanhydrous sodium sulfate, filtered and evaporated. The crude product wasobtained after removal of the solvent. Upon purification over silica gelusing 3% MeOH/DCM compound 75 (8.46 g, 87%) was obtained as white foamysolid.

¹H NMR (CDCl₃, 400 MHz): δ 7.18-7.4 (m, 14H), 6.8 (m, 4H), 5.1 (s, 2H),5.0 (m, 1H), 4.54 (m, 1H), 4.18 (m, 2H), 3.78 (s, 6H), 3.6 (m, 2H), 3.14(m, 1H), 2.02 (m, 1H), 1.74 (m, 1H). (Also observed minor rotamer due toamide bond).

¹³C NMR (CDCl₃, 100 MHz): δ 158.81, 158.59, 147.52, 145.18, 139.65,136.64, 136.31, 130.17, 129.33, 128.76, 128.58, 128.37, 128.28, 128.18,128.05, 127.96, 127.28, 126.93, 113.35, 113.24, 86.1, 81.62, 69.93,67.66, 67.16, 66.81, 55.45, 55.39, 37.64.

Synthesis of4-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-pyrrolidin-3-ol (76)

Compound 75 (8.25 g, 14 9 mmol) was dissolved in methanol (20 mL) andpurged with argon. To the solution was added 10% palladium on carbon(0.825 g). The flask was purged with hydrogen 2 times and stirredfurther at room temperature under hydrogen atmosphere for 3 h.

The disappearance of the starting material was confirmed by the TLC. Thereaction mixture was filtered through a pad of Celite and washed withmethanol. The combined organic layer was concentrated under reducedpressure to afford amine 76 (6.12 g, 98%) as white solid. This was usedas such for the next step.

¹H NMR (DMSO-d₆, 400 MHz): δ 8.3 (s, 1H), 7.38 (m, 2H), 7.22 (m, 7H),6.84 (m, 4H), 4.2 (m, 1H), 3.7 (s, 6H), 3.6 (m, 1H), 3.0 (m, 3H), 2.8(m, 1H), 1.74 (m, 1H), 1.5 (m, 1H).

¹³C NMR (DMSO-d₆, 100 MHz): δ 165.02, 158.06, 144.90, 135.55, 129.74,127.81, 127.72, 126.67, 113.15, 85.58, 69.6, 59.76, 56.81, 55.02, 53.62.

Synthesis of2-(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-isoindole-1,3-dione(77)

Referring to scheme 18, compound 76 (4.2 g, 10 mmol) was dissolved inanhydrous dichloromethane (25 mL) and cooled to 0° C. To the solutionwere added triethylamine (1.01 g, 1.4 mL, 10 mmol) and ester 43 (4.3 g,10 mmol) successively. The reaction temperature was brought to ambienttemperature and stirred further for 2 h. The completion of the reactionwas ascertained by TLC (10% MeOH/CHCl₃). The reaction mixture wasdiluted with DCM (100 mL) and washed with 10% NaOH aolution. The organiclayer was washed with brine, water and dried over anhydrous sodiumsulfate and filtered. The crude product was obtained by evaporating thesolvent under the vacuum. Compound 77 (5.4 g, 81%) was obtained as awhite foamy solid after column chromatography over silica gel using 4%MeOH/DCM.

¹H NMR (DMSO-d₆, 400 MHz): δ 7.84 (m, 4H), 7.28 (m, 4H), 7.18 (m, 5H),6.86 (m, 4H), 4.98 (d, —OH, D₂O exchangeable), 4.38 (m, 1H), 4.3 (m,1H), 3.72 (s, 6H), 3.53 (m, 3H), 3.3 (m, 1H), 3.14 (m, 1H), 2.98 (m,2H), 2.2 (m, 2H), 2.0 (m, 2H), 1.44-1.62 (m, 2H), 1.3 (m, 2H), 1.13 (m,1H)

¹³C NMR (DMSO-d₆, 100 MHz): δ 172.78, 172.11, 168.74, 168.67, 158.74,158.55, 158.54, 145.24, 144.72, 136.46, 136.27, 137.87, 135.84, 134.18,134.13, 134.09, 132.3, 132.27, 130.18, 130.11, 129.33, 128.22, 128.20,128.08, 128.03, 127.93, 127.12, 126.89, 123.41, 123.38, 113.35, 113.2,86.7, 86.06, 70.7, 69.46, 65.51, 63.67, 56.61, 56.0, 55.9, 55.42, 55.36,54.2, 38.44, 38.0, 37.98, 36.9, 35.0, 33.4, 28.6, 28.5, 28.4, 26.79,26.71, 25.0, 24.6, 24.5.

Pyrrolidine-Phthalimido Phosphoramidite (78)

Compound 77 (1.5 g, 2.26 mmol) was coevaporated with anhydrous toluene(25 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.127 g,1.13 mmol) was added and the mixture was dried over P₂O₅ in a vacuumoven for overnight at 40° C. The reaction mixture was dissolved inacetonitrile/dichloroethane (10 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.362 g, 1.49mL, 4.52 mmol) was added. The reaction mixture was stirred at ambienttemperature for overnight. The completion of the reaction wasascertained by TLC(R_(f)=0.4 in 1:1 ethyl acetate:hexane). The reactionmixture was concentrated undervacuum and the residue was dissolved inethyl acetate (100 mL). The organic layer was washed with 5% NaHCO₃ (100mL) and brine (100 mL). The organic layer was dried over anhydrousNa₂SO₄ filtered and concentrated under reduced pressure. The residue waspurified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to affordcompound 78 as white solid (1.66 g, 85%).

¹H NMR (400 MHz, C₆D₆): δ 7.62 (m, 2H), 7.42 (m, 6H), 7.22 (t, 2H), 7.08(m, 1H), 6.88 (dd, 2H), 6.78 (m, 4H), 4.66 (m, 1H), 4.56 (m, 1H), 3.72(m, 1H), 3.5 (m, 5H), 3.3 (m, 7H), 3.22 (m, 1H), 2.1 (m, 5H), 1.74 (m,4H), 1.56 (m, 2H), 1.26 (m, 2H), 1.1 (m, 13H).

³¹P NMR (161.82 MHz, C₆D₆): δ 146.3, 146.2, 145.98, 145.8, 145.63, 145.4(multiple peaks due mixer of diastereomer and Rotamers observed afterdue to amide bond at the ring)

¹³C NMR (100 MHz, C₆D₆): δ 171.03, 170.08, 167.98, 159.23, 159.0, 146.1,136.76, 136.69, 136.64, 136.27, 133.35, 132.7, 130.59, 130.54, 130.46,128.65, 128.56, 127.55, 126.97, 128.24, 128.0, 127.7, 122.84, 113.62,113.53, 113.51, 86.57, 86.51, 72.67, 72.5, 72.33, 64.48, 58.59, 58.46,58.41, 58.28, 57.77, 56.03, 55.97, 54.81, 54.73, 43.47, 43.35, 37.87,36.42, 36.32, 34.94, 34.88, 33.37, 28.77, 26.94, 24.67, 24.6, 24.51,20.10, 20.04, 19.98.

Synthesis of Succinic acidmono-{4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-[6-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-hexanoyl]-pyrrolidin-3-yl}ester(79)

Referring to scheme 19, Compound 77 (2 g, 3 mmol) was mixed withsuccinic anhydride (0.600 g, 6 mmol) and DMAP (0.366 g, 3 mmol) anddried in a vacuum at 40° C. overnight. The mixture was dissolved inanhydrous dichloromethane (5 mL), triethylamine (0.913 g, 1.25 mL, 9mmol) was added and the solution was stirred at room temperature underargon atmosphere for 4 h. It was then diluted with dichloromethane (50mL) and washed with ice cold aqueous citric acid (5% wt., 50 mL) andwater (2×50 mL). The organic phase was dried over anhydrous sodiumsulfate and concentrated to dryness. The crude product was purified bycolumn chromatography using 6% MeOH/DCM to afford compound 79 as whitesolid (2.05 g, 89% yield; R_(f)=0.4 in 10% MeOH/CHCl₃).

¹H NMR (400 MHz, DMSO-d₆): δ 7.8 (m, 4H), 7.26 (m, 4H), 7.14 (m, 5H),6.83 (m, 4H), 4.92 (d, —OH, D₂O exchangeable), 4.38 (m, 1H), 4.1 (m,1H), 3.68 (s, 6H), 3.52 (m, 2H), 3.3 (m, 2H), 3.1 (m, 1H), 2.95 (m, 1H),2.18 (m, 6H), 1.98 (m, 2H), 1.44-1.58 (m, 4H), 1.26 (m, 2H)

¹³C NMR (100 MHz, DMSO-d₆): δ 170.78, 167.94, 158.07, 157.96, 145.07,135.86, 135.44, 134.36, 131.61, 129.6, 127.78, 127.57, 126.58, 122.99,113.10, 85.10, 68.56, 54.98, 37.28. 27.87, 26.01, 24.03.

Synthesis of Phthalimido-Pyrrolidine Immobilized on a Solid Support (80)

Succinate 79 (0.900 g, 1.17 mmol) was dissolved in dichloroethane:ACN(1:1, 5 mL). To that solution DMAP (0.144 g, 1.17 mmol) was added.2,2′-Dithio-bis(5-nitropyridine) (0.360 g, 1.17 mmol) inacetonitrile/dichloroethane (3:1, 5 mL) was added successively. To theresulting solution triphenylphosphine (0.306 g, 1.17 mmol) inacetonitrile (2.5 ml) was added. The reaction mixture turned brightorange in color. The solution was agitated briefly using wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (3.5 g, 155 μm/g)was added. The suspension was agitated for 4 h. The CPG was filteredthrough a sintered funnel and washed with acetonitrile, dichloromethaneand ether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The loading capacity of the CPG 80 was measured bytaking UV measurement. (87 μM/g)

Synthesis of Extended Steroid Conjugates with Hydroxyl-Prolinol Linker

Synthesis of4-(3-Hydroxy-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-17-yl)-pentanoicacid dioctadecylamide (82)

Lithocholic acid (81) (7.1 g, 18 8 mmol) was sissolved in anhydroustetrahydrofuran (60 mL). Isobutylchloroformate (2.6 g, 2.6 mL, 18 8mmol) was added followed by the addition of triethylamine (3.84 g, 5.3mL, 38 mmol) and dioctadecylamine (9.8 g, 18.77 g). The reaction mixturewas brought to ambient temperature and allowed to stir over night. Thereaction mixture was concentrated under vacuum, and the residue wasdissolved in dichloromethane (250 mL). The organic layer was washed with5% sodium bicarbonate, 3% aqueous HCl and water. After drying overanhydrous sodium sulfate, the solvent was removed under reduced pressureto afford amide 82 (15.5 g) in 93% yield. This was used as such for thenext step.

¹H NMR (400 MHz, CDCl₃): δ 3.84 (d, —OH, D₂O exchangeable), 3.64 (m,1H), 3.16-3.34 (m, 4H), 2.32 (m, 1H), 2.18 (m, 1H), 1.22-1.98 (m, 83H),0.84-1.18 (m, 17H), 0.64 (s, 3H)

¹³C NMR (100 MHz, CDCl₃): δ 173.3, 72.1, 71.04, 56.7, 56.3, 56.19, 48.2,46.07, 42.96, 42.32, 40.65, 40.41, 36.70, 36.07, 35.87, 35.57, 34.79,32.13, 31.92, 30.77, 30.37, 29.83, 29.8, 29.78, 29.72, 29.68, 29.4,28.47, 28.3, 28.03, 27.41, 27.29, 27.13, 27.09, 26.63, 24.45, 23.59,22.9, 21.05, 19.41, 18.76, 18.48, 14.32, 12.28.

Synthesis of Carbonic acid17-(3-dioctadecylcarbamoyl-1-methyl-propyl)-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-3-ylester 2,5-dioxo-pyrrolidin-1-yl ester (83)

Referring to scheme 20, amide 82 (15.5 g, 17.6 mmol) was dissolved inanhydrous dichloromethane (150 mL). To the solution were addeddisuccinimidyl carbonate (6.76 g, 26.4 mmol), triethylamine (10 mL) andacetonitrile (50 mL). The reaction mixture was stirred at roomtemperature under argon for 6 h and then evaporated dryness. The residuewas dissolved in dichloromethane (300 mL). It was washed with saturatedNaHCO₃ aqueous solution (3×100 mL). The organic layer was dried overNa₂SO₄, filtered and evaporated to dryness. Compound 83 (12.3 g, 71%)was obtained as colorless powder after drying in high vacuum, which wasdirectly used for the next step without further purification.

¹H NMR (400 MHz, CDCl₃): δ 4.7 (m, 1H), 3.15-3.34 (m, 4H), 2.82 (s, 4H),2.32 (m, 1H), 2.16 (m, 1H), 1.66-2.0 (m, 10H), 1.2-1.58 (m, 78H),0.86-1.12 (14H), 0.64 (s, 3H).

¹³C NMR (100 MHz, CDCl₃): δ 173.34, 168.98, 151.08, 83.17, 71.37, 56.58,56.31, 48.21, 46.06, 42.91, 42.09, 40.61, 40.26, 35.95, 35.81, 35.33,34.96, 34.75, 34.69, 32.10, 31.19, 31.88, 29.89, 29.85, 29.79, 29.54,28.41, 28.27, 27.96, 27.26, 27.09, 27.06, 26.42, 25.82, 25.77, 25.73,25.66, 25.6, 24.38, 23.56, 23.34, 22.87, 21.02, 20.35, 19.37, 18.83,18.73, 18.53, 14.3, 12.25.

Synthesis of(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(3-dioctadecylcarbamoyl-1-methyl-propyl)-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-3-ylester (LI)

Amine 5 (4.22 g, 7 9 mmol) was dissolved in anhydrous dichloromethane(25 mL) and cooled to 0° C. To the solution were added pyridine (10 mL)and compound 83 (8.1 g, 7 9 mmol) successively. The reaction temperaturewas brought to ambient temperature and stirred further for 3 h. Thecompletion of the reaction was ascertained by TLC (EtOAc, R_(f)=0.8).The reaction mixture was diluted with dichloromethane and washed withsaturated NaHCO₃, water followed by brine. The organic layer was driedover sodium sulfate, filtered and concentrated under vacuum to affordthe crude product. Compound 12 (8.8 g, 77%) was obtained as a whitesolid after column chromatography over silica gel.

¹H NMR (400 MHz, DMSO-d₆): δ 7.2-7.38 (m, 9H), 6.76 (m, 4H), 4.0 (m,2H), 3.72 (s, 6H), 3-3.18 (m, 3H), 2.96 (m, 2H), 2.5-2.6 (m, 3H),2.12-2.38 (m, 6H), 1.22-1.98 (m, 89H), 0.84-1.18 (m, 23H), 0.64 (s, 3H)

¹³C NMR (100 MHz, DMSO-d₆): δ 171.89, 171.38, 158.74, 158.56, 156.70,156.6, 145.28, 144.77, 136.49, 136.33, 135.89, 135.8, 130.19, 130.15,128.25, 128.20, 128.09, 127.94, 127.13, 126.90, 113.37, 113.21, 72.1,71.04, 56.7, 56.3, 56.19, 48.2, 46.07, 42.96, 42.32, 40.65, 40.41,36.70, 36.07, 35.87, 35.57, 34.79, 32.13, 31.92, 30.77, 30.37, 29.83,29.8, 29.78, 29.72, 29.68, 29.4, 28.47, 28.3, 28.03, 27.41, 27.29,27.13, 27.09, 26.63, 24.45, 23.59, 22.9, 21.05, 19.41, 18.76, 18.48,14.32, 12.28.

Synthesis of Extended Steroid Conjugates Phosphoramidite withHydroxyl-Prolinol Linker (85)

Compound 84 (5.8 g, 4 mmol) was coevaporated with anhydrous toluene (50mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.225 g, 2mmol) was added and the mixture was dried over P₂O₅ in a vacuum oven forovernight at 40° C. The reaction mixture was dissolved indichloromethane (25 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.8 g, 1.97 mL,6 mmol) was added. The reaction mixture was stirred at ambienttemperature for overnight. The completion of the reaction wasascertained by TLC(R_(f)=0.7 in 1:1 ethyl acetate:hexane). The reactionmixture was diluted with dichloromethane (100 mL) and washed with 5%NaHCO₃ (100 mL) and brine (100 mL). The organic layer was dried overanhydrous Na₂SO₄ filtered and concentrated under reduced pressure. Theresidue was purified over silica gel (50:49:1,EtOAc:Hexane:triethlyamine) to afford 85 as white solid (5.45 g, 83%).

¹H NMR (400 MHz, C₆D₆): δ 7.62 (m, 2H), 7.46 (m, 4H), 7.24 (m, 2H), 7.08(m, 1H), 6.8 (m, 4H), 4.9 (m, 1H), 4.6 (m, 2H), 3.74 (m, 1H), 3.5 (m,3H), 3.4 (m, 2H), 3.36 (2s, 6H), 3-3.22 (m, 4H), 0.8-2.4 (m, 133H), 0.62(s, 3H)

³¹P NMR (161.82 MHz, CDCl₃): δ 148.26 (high in integration), 148.01,147.6 (due to rotamer

¹³C NMR (100 MHz, CDCl₃): δ 171.79, 171.61, 158.75, 158.58, 156.59,145.31, 144.77, 136.47, 136.35, 136.31, 135.86, 130.22, 130.19, 128.28,128.20, 128.11, 127.95, 127.15, 126.91, 113.39, 113.24, 86.11, 71.98,70.81, 70.69, 72.93, 72.2, 71.98, 70.81, 70.81, 70.69, 64.37, 63.92,58.55, 58.35, 58.36, 58.16, 59.57, 55.86, 55.44, 55.39, 46.31, 44.70,44.65, 43.36, 43.34, 41.08, 35.08, 33.45, 32.13, 30.23, 29.92, 29.88,29.72, 29.58, 26.32, 26.26, 24.85, 24.78, 24.68, 22.9, 20.58, 14.34.

Synthesis of Succinic acidmono-(5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(3-dioctadecylcarbamoyl-1-methyl-propyl)-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-3-yl)ester (86)

Referring to scheme 21, Compound 84 (1.44 g, 1 mmol) was mixed withsuccinic anhydride (0.15 g, 1.5 mmol) and DMAP (0.0122 g, 0.1 mmol) anddried in a vacuum at 40° C. overnight. The mixture was dissolved inanhydrous dichloromethane (5 mL), triethylamine (0.101 g, 0.14 mL, 1mmol) was added and the solution was stirred at room temperature underargon atmosphere for 16 h. It was then diluted with dichloromethane (50mL) and washed with ice cold aqueous citric acid (5% wt., 25 mL) andwater (2×25 mL). The organic phase was dried over anhydrous sodiumsulfate and concentrated to dryness. The crude product was purified bycolumn chromatography to afford compound 86 as white solid (1.1 g, 71%yield; R_(f)=0.5 in 10% MeOH/CHCl₃).

¹H NMR (400 MHz, DMSO-d₆): δ 7.62 (m, 2H), 7.46 (m, 4H), 7.24 (m, 2H),7.08 (m, 1H), 6.8 (m, 4H), 4.9 (m, 1H), 4.6 (m, 2H), 3.74 (m, 1H), 3.5(m, 3H), 3.4 (m, 2H), 3.36 (2s, 6H), 2.82 (s, 4H), 2.32 (m, 1H), 2.16(m, 1H), 1.66-2.0 (m, 10H), 1.2-1.58 (m, 78H), 0.86-1.12 (14H), 0.64 (s,3H).

¹³C NMR (100 MHz, DMSO-d₆): δ 176.59, 172.22, 158.78, 158.62, 145.16,139.8, 136.39, 136.22, 130.18, 130.14, 128.23, 128.0, 126.97, 122.91,113.28, 72.1, 71.04, 56.7, 56.3, 56.19, 48.2, 46.07, 42.96, 42.32,40.65, 40.41, 36.70, 36.07, 35.87, 35.57, 34.79, 32.13, 31.92, 30.77,30.37, 29.83, 29.8, 29.78, 29.72, 29.68, 29.4, 28.47, 28.3, 28.03,27.41, 27.29, 27.13, 27.09, 26.63, 24.45, 23.59, 22.9, 21.05, 19.41,18.76, 18.48, 14.32, 12.28.

Synthesis of Extended Steroid Conjugates Immobilized on Solid Supportwith Hydroxyl-Prolinol Linker (87)

Succinate 86 (1 g, 0.649 mmol) was dissolved in dichloroethane (3 mL).To that solution DMAP (0.079 g, 0.649 mmol) was added.2,2′-Dithio-bis(5-nitropyridine) (0.202 g, 0.649 mmol) inacetonitrile/dichloroethane (3:1, 3 mL) was added successively. To theresulting solution triphenylphosphine (0.17 g, 0.65 mmol) inacetonitrile (1.5 ml) was added. The reaction mixture turned brightorange in color. The solution was agitated briefly using wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (4 g, 155 μm/g)was added. The suspension was agitated for 16 h. The CPG was filteredthrough a sintered funnel and washed with acetonitrile, dichloromethaneand ether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The loading capacity of the CPG was measured bytaking UV measurement. (62 μM/g).

Synthesis of Extended Steroid Conjugates with Hydroxyl-Prolinol Linker

Synthesis of4-(3-Hydroxy-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-17-yl)-pentanoicacid octadecylamide (88)

Lithocholic acid (81) (9.78 g, 26 mmol) was sissolved in anhydroustetrahydrofuran (60 mL). Isobutylchloroformate (3.55 g, 3.55 mL, 26mmol) was added followed by the addition of triethylamine (5.26 g, 7.25mL, 52 mmol) and dioctadecylamine (7 g, 26 g). The reaction mixture wasbrought to ambient temperature and allowed to stir over night. Thereaction mixture was concentrated under vacuum, and the residue wasdissolved in dichloromethane (250 mL). The organic layer was washed with5% sodium bicarbonate, 3% aqueous HCl and water. After drying overanhydrous sodium sulfate, the solvent was removed under reduced pressureto afford amide 88 (14.5 g) in 89% yield. This was used as such for thenext step.

Synthesis of Carbonic acid10,13-dimethyl-17-(1-methyl-3-octadecylcarbamoyl-propyl)-hexadecahydro-cyclopenta[a]phenanthren-3-ylester 2,5-dioxo-pyrrolidin-1-yl ester (89)

Referring to scheme 22, amide 88 (14.5 g, 23 mmol) was dissolved inanhydrous dichloromethane (150 mL). To the solution were addeddisuccinimidyl carbonate (8.87 g, 34 mmol), triethylamine (15 mL) andacetonitrile (50 mL). The reaction mixture was stirred at roomtemperature under argon for 6 h and then evaporated dryness. The residuewas dissolved in dichloromethane (300 mL). It was washed with saturatedNaHCO₃ aqueous solution (3×100 mL). The organic layer was dried overNa₂SO₄, filtered and evaporated to dryness. Compound 89 (14.3 g, 81%)was obtained as colorless powder after drying in high vacuum, which wasdirectly used for the next step without further purification.

Synthesis of(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid10,13-dimethyl-17-(1-methyl-3-octadecylcarbamoyl-propyl)-hexadecahydro-cyclopenta[a]phenanthren-3-ylester (90)

Amine 5 (4.22 g, 7 9 mmol) is dissolved in anhydrous dichloromethane (25mL) and cooled to 0° C. To the solution are added pyridine (10 mL) andcompound 89 (9.3 g, 7 9 mmol) successively. The reaction temperature wasbrought to ambient temperature and stirred further. The completion ofthe reaction is ascertained by TLC (EtOAc). The reaction mixture isdiluted with dichloromethane and washed with saturated NaHCO₃, waterfollowed by brine. The organic layer is dried over sodium sulfate,filtered and concentrated under vacuum to afford the crude product.Compound 90 is obtained as a white solid after column chromatographyover silica gel.

Synthesis of Extended Steroid Conjugates Phosphoramidite withHydroxyl-Prolinol Linker (91)

Compound 90 (4.75 g, 4 mmol) is coevaporated with anhydrous toluene (50mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.225 g, 2mmol) is added and the mixture is dried over P₂O₅ in a vacuum oven forovernight at 40° C. The reaction mixture is dissolved in dichloromethane(25 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.8g, 1.97 mL, 6 mmol) is added. The reaction mixture is stirred at ambienttemperature for overnight. The completion of the reaction is ascertainedby TLC (1:1 ethyl acetate:hexane). The reaction mixture is diluted withdichloromethane (100 mL) and washed with 5% NaHCO₃ (100 mL) and brine(100 mL). The organic layer is dried over anhydrous Na₂SO₄ filtered andconcentrated under reduced pressure. The residue was purified oversilica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 91.

Synthesis of(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid10,13-dimethyl-17-(1-methyl-3-octadecylcarbamoyl-propyl)-hexadecahydro-cyclopenta[a]phenanthren-3-ylester (92)

Referring to scheme 23, Compound 90 (1.185 g, 1 mmol) is mixed withsuccinic anhydride (0.15 g, 1.5 mmol) and DMAP (0.0122 g, 0.1 mmol) anddried in a vacuum at 40° C. overnight. The mixture is dissolved inanhydrous dichloromethane (5 mL), triethylamine (0.101 g, 0.14 mL, 1mmol) is added and the solution is allowed to stir at room temperatureunder argon atmosphere for 16 h. It is then diluted with dichloromethane(50 mL) and washed with ice cold aqueous citric acid (5% wt., 25 mL) andwater (2×25 mL). The organic phase is dried over anhydrous sodiumsulfate and concentrated to dryness. The crude product was purified bycolumn chromatography to afford compound 92.

Synthesis of Extended Steroid Conjugates Immobilized on Solid Supportwith Hydroxyl-Prolinol Linker (93)

Succinate 92 (0.833 g, 0.649 mmol) is dissolved in dichloroethane (3mL). To that solution DMAP (0.079 g, 0.649 mmol) is added.2,2′-Dithio-bis(5-nitropyridine) (0.202 g, 0.649 mmol) inacetonitrile/dichloroethane (3:1, 3 mL) is added successively. To theresulting solution triphenylphosphine (0.17 g, 0.65 mmol) inacetonitrile (1.5 ml) is added. The reaction mixture turned brightorange in color. The solution is agitated briefly using wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (2.5 g, 155 μm/g)isadded. The suspension is agitated further. The CPG is filtered througha sintered funnel and washed with acetonitrile, dichloromethane andether successively. Unreacted amino groups are masked using aceticanhydride/pyridine. The loading capacity of the CPG is measured bytaking UV measurement.

Synthesis of Dimethylamino Phosphoramidite with Hydroxyl-Prolinol Linker1-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-dimethylamino-hexan-1-one(94)

Hydrochloride salt of 5-dimethylamino-pentanoic acid (1.95 g, 10 mmol)is suspended in anhydrous pyridine (50 mL). To the suspension is addeddiisopropylcarbodiimide (1.262 g, 1.55 mL, 10 mmol) followed by amine 38(4.2 g, 10 mmol). The stirring is allowed to continue for 16 h. Thereaction mixture is concentrated under vacuum, to the residue ethylacetate is added and washed with, 5% NaHCO₃ solution, brine and water.After drying over anhydrous sodium sulfate the solvent is removed toafford crude product. DMT-alcohol 94 is obtained after purification oversilica gel.

Synthesis of Dimethylamino Phosphoramidite with Hydroxyl-Prolinol Linker(95)

Compound 94 (2.35 g, 4.2 mmol) is coevaporated with anhydrous toluene(25 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.238 g,2.1 mmol) is added and the mixture was dried over P₂O₅ in a vacuum ovenfor overnight at 40° C. The reaction mixture is dissolved indichloromethane (25 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.9 g, 2.1 mL,6.3 mmol) is added. The reaction mixture was stirred at ambienttemperature for overnight. The completion of the reaction isascertainedby TLC (1:1 ethyl acetate:hexane). The reaction mixture is diluted withdichloromethane (50 mL) and washed with 5% NaHCO₃ (50 mL) and brine (50mL). The organic layer is dried over anhydrous Na₂SO₄ filtered andconcentrated under reduced pressure. The residue was purified oversilica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 95.

Synthesis of Succinic acidmono-[5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-(6-dimethylamino-hexanoyl)-pyrrolidin-3-yl]ester(1

Referring to scheme 25, Compound 94 (1.2 g, 2 mmol) is mixed withsuccinic anhydride (0.200 g, 2 mmol) and DMAP (0.244 g, 13 mmol) anddried in a vacuum at 40° C. overnight. The mixture is dissolved inanhydrous dichloromethane (5 mL), triethylamine (0.476 g, 0.64 mL, 4mmol) is added and the solution stirred at room temperature under argonatmosphere for 16 h. It is then diluted with dichloromethane (100 mL)and washed with ice cold aqueous citric acid (5% wt., 100 mL) and water(2×100 mL). The organic phase is dried over anhydrous sodium sulfate andconcentrated to dryness. The crude product is purified by columnchromatography to afford compound 96.

Synthesis of Dimethylamino Immobilized Solid Support withHydroxyl-Prolinol Linker (97)

Succinate 96 (1 g, 1.5 mmol) is dissolved in dichloroethane (7 mL). Tothat solution DMAP (0.183 g, 1.5 mmol) is added.2,2′-Dithio-bis(5-nitropyridine) (0.470 g, 1 5 mmol) inacetonitrile/dichloroethane (3:1, 7 mL) is added successively. To theresulting solution triphenylphosphine (0.395 g, 1.5 mmol) inacetonitrile (3 ml) is added. The reaction mixture turned bright orangein color. The solution is agitated briefly using wrist-action shaker (5mins). Long chain alkyl amine-CPG (LCAA-CPG) (4 g, 155 μm/g) is added.The suspension was agitated for 4 h. The CPG is filtered through asintered funnel and washed with acetonitrile, dichloromethane and ethersuccessively. Unreacted amino groups are masked using aceticanhydride/pyridine. The loading capacity of the CPG is measured bytaking UV measurement.

Synthesis of Nalidixic Phosphoramidite with Hydroxyl-Prolinol Linker

1-Ethyl-7-methyl-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid(6-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-amide(99)

Nalidixic acid (2.32 g, 10 mmol) is suspended in anhydrous pyridine (50mL). To the suspension is added diisopropylcarbodiimide (1.262 g, 1.55mL, 10 mmol) followed by amine 5 (5.32 g, 10 mmol). The stirring isallowed to continue for 16 h. The reaction mixture is concentrated undervacuum, to the residue ethyl acetate is added and washed with, 5% NaHCO₃solution, brine and water. After drying over anhydrous sodium sulfatethe solvent is removed to afford crude product. DMT-alcohol 99 isobtained after purification over silica gel.

Synthesis of Nalidixic Phosphoramidite with Hydroxyl-Prolinol Linker(100)

Compound 99 (3.12 g, 4.2 mmol) is coevaporated with anhydrous toluene(25 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.238 g,2.1 mmol) is added and the mixture was dried over P₂O₅ in a vacuum ovenfor overnight at 40° C. The reaction mixture is dissolved indichloromethane (25 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.9 g, 2.1 mL,6.3 mmol) is added. The reaction mixture was stirred at ambienttemperature for overnight. The completion of the reaction isascertainedby TLC (1:1 ethyl acetate:hexane). The reaction mixture is diluted withdichloromethane (50 mL) and washed with 5% NaHCO₃ (50 mL) and brine (50mL). The organic layer is dried over anhydrous Na₂SO₄ filtered andconcentrated under reduced pressure. The residue was purified oversilica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 100.

Synthesis of Succinic acidmono-(5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[(1-ethyl-7-methyl-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carbonyl)-amino]hexanoyl}-pyrrolidin-3-yl)ester (101)

Referring to scheme 27, Compound 99 (1.48 g, 2 mmol) is mixed withsuccinic anhydride (0.200 g, 2 mmol) and DMAP (0.244 g, 13 mmol) anddried in a vacuum at 40° C. overnight. The mixture is dissolved inanhydrous dichloromethane (5 mL), triethylamine (0.606 g, 0.96 mL, 6mmol) is added and the solution stirred at room temperature under argonatmosphere for 16 h. It is then diluted with dichloromethane (100 mL)and washed with ice cold aqueous citric acid (5% wt., 100 mL) and water(2×100 mL). The organic phase is dried over anhydrous sodium sulfate andconcentrated to dryness. The crude product is purified by columnchromatography to afford compound 101.

Synthesis of Nalidixic Immobilized Solid Support with Hydroxyl-ProlinolLinker (102)

Succinate 100 (1.26 g, 1.5 mmol) is dissolved in dichloroethane (7 mL).To that solution DMAP (0.183 g, 1.5 mmol) is added.2,2′-Dithio-bis(5-nitropyridine) (0.470 g, 1 5 mmol) inacetonitrile/dichloroethane (3:1, 7 mL) is added successively. To theresulting solution triphenylphosphine (0.395 g, 1.5 mmol) inacetonitrile (3 ml) is added. The reaction mixture turned bright orangein color. The solution is agitated briefly using wrist-action shaker (5mins). Long chain alkyl amine-CPG (LCAA-CPG) (4 g, 155 μm/g) is added.The suspension was agitated for 4 h. The CPG is filtered through asintered funnel and washed with acetonitrile, dichloromethane and ethersuccessively. Unreacted amino groups are masked using aceticanhydride/pyridine. The loading capacity of the CPG is measured bytaking UV measurement.

Diosgenein Phosphoramidite with Hydroxyl-Prolinol Linker

Diosgenin Succinimidyl Carbamate (104)

Referring to scheme 28, diosgenin (6.9 g, 16.74 mmol) is dissolved inanhydrous dichloromethane (150 mL). To the solution are addeddisuccinimidyl carbonate (6.4 g, 25.1 mmol), triethylamine (10 mL) andacetonitrile (50 mL). The reaction mixture is stirred at roomtemperature under argon for 6 h and then evaporated dryness. The residueiss dissolved in dichloromethane (300 mL). It is washed with saturatedNaHCO₃ aqueous solution (3×100 mL). The organic layer is dried overNa₂SO₄, filtered and evaporated to dryness. Compound 104 is obtained ascolorless powder after drying in high vacuum, which is directly used forthe next step without further purification.

Synthesis of Diosgenin DMT-Alcohol 105

Amine 5 (10.5 g, 19 7 mmol) is dissolved in anhydrous dichloromethane(50 mL) and cooled to 0° C. To the solution were added pyridine (10 mL)and compound 104 (9.62 g, 17.3 mmol) successively. The reactiontemperature is brought to ambient temperature and stirred further for 3h. The completion of the reaction is ascertained by TLC (10%MeOH/CHCl₃). The reaction mixture is diluted with dichloromethane andwashed with saturated NaHCO₃, water followed by brine. The organic layeris dried over sodium sulfate, filtered and concentrated under vacuum toafford the crude product. Compound 105 is obtained as a white solidafter column chromatography over silica gel.

Synthesis of Diosgenin Phosphoramidite with Hydroxyl-Prolinol Linker(106)

Compound 105 (4.1 g, 4.2 mmol) is coevaporated with anhydrous toluene(25 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.238 g,2.1 mmol) is added and the mixture was dried over P₂O₅ in a vacuum ovenfor overnight at 40° C. The reaction mixture is dissolved indichloromethane (25 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.9 g, 2.1 mL,6.3 mmol) is added. The reaction mixture was stirred at ambienttemperature for overnight. The completion of the reaction isascertainedby TLC (1:1 ethyl acetate:hexane). The reaction mixture is diluted withdichloromethane (50 mL) and washed with 5% NaHCO₃ (50 mL) and brine (50mL). The organic layer is dried over anhydrous Na₂SO₄ filtered andconcentrated under reduced pressure. The residue was purified oversilica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 106.

Synthesis of Diosgenin-Hydroxy-Prolinol Succinate 107

Referring to scheme 29, Compound 105 (1.95 g, 2 mmol) is mixed withsuccinic anhydride (0.200 g, 2 mmol) and DMAP (0.244 g, 13 mmol) anddried in a vacuum at 40° C. overnight. The mixture is dissolved inanhydrous dichloromethane (5 mL), triethylamine (0.676 g, 0.96 mL, 6mmol) is added and the solution stirred at room temperature under argonatmosphere for 16 h. It is then diluted with dichloromethane (100 mL)and washed with ice cold aqueous citric acid (5% wt., 100 mL) and water(2×100 mL). The organic phase is dried over anhydrous sodium sulfate andconcentrated to dryness. The crude product is purified by columnchromatography to afford compound 107.

Synthesis of Diosgenin Immobilized Solid Support with Hydroxyl-ProlinolLinker (108)

Succinate 107 (1.61 g, 1.5 mmol) is dissolved in dichloroethane (7 mL).To that solution DMAP (0.183 g, 1.5 mmol) is added.2,2′-Dithio-bis(5-nitropyridine) (0.470 g, 1 5 mmol) inacetonitrile/dichloroethane (3:1, 7 mL) is added successively. To theresulting solution triphenylphosphine (0.395 g, 1.5 mmol) inacetonitrile (3 ml) is added. The reaction mixture turned bright orangein color. The solution is agitated briefly using wrist-action shaker (5mins). Long chain alkyl amine-CPG (LCAA-CPG) (4 g, 155 μm/g) is added.The suspension was agitated for 4 h. The CPG is filtered through asintered funnel and washed with acetonitrile, dichloromethane and ethersuccessively. Unreacted amino groups are masked using aceticanhydride/pyridine. The loading capacity of the CPG is measured bytaking UV measurement.

Epifriedelanol Phosphoramidite with Hydroxyl-Prolinol Linker

Epifriedelanol Succinimidyl Carbamate (110)

Referring to scheme 30, Epifriedelanol (7.2 g, 16.74 mmol) is dissolvedin anhydrous dichloromethane (150 mL). To the solution are addeddisuccinimidyl carbonate (6.4 g, 25.1 mmol), triethylamine (10 mL) andacetonitrile (50 mL). The reaction mixture is stirred at roomtemperature under argon for 6 h and then evaporated dryness. The residueiss dissolved in dichloromethane (300 mL). It is washed with saturatedNaHCO₃ aqueous solution (3×100 mL). The organic layer is dried overNa₂SO₄, filtered and evaporated to dryness. Compound 110 is obtained ascolorless powder after drying in high vacuum, which is directly used forthe next step without further purification.

Synthesis of Epifriedelanol DMT-Alcohol 111

Amine 5 (10.5 g, 19 7 mmol) is dissolved in anhydrous dichloromethane(50 mL) and cooled to 0° C. To the solution were added pyridine (10 mL)and compound 110 (9.85 g, 17.3 mmol) successively. The reactiontemperature is brought to ambient temperature and stirred further for 3h. The completion of the reaction is ascertained by TLC (10%MeOH/CHCl₃). The reaction mixture is diluted with dichloromethane andwashed with saturated NaHCO₃, water followed by brine. The organic layeris dried over sodium sulfate, filtered and concentrated under vacuum toafford the crude product. Compound III is obtained as a white solidafter column chromatography over silica gel.

Synthesis of Epifriedelanol Phosphoramidite with Hydroxyl-ProlinolLinker (12)

Compound III (4.14 g, 4.2 mmol) is coevaporated with anhydrous toluene(25 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.238 g,2.1 mmol) is added and the mixture was dried over P₂O₅ in a vacuum ovenfor overnight at 40° C. The reaction mixture is dissolved indichloromethane (25 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.9 g, 2.1 mL,6.3 mmol) is added. The reaction mixture was stirred at ambienttemperature for overnight. The completion of the reaction isascertainedby TLC (1:1 ethyl acetate:hexane). The reaction mixture is diluted withdichloromethane (50 mL) and washed with 5% NaHCO₃ (50 mL) and brine (50mL). The organic layer is dried over anhydrous Na₂SO₄ filtered andconcentrated under reduced pressure. The residue was purified oversilica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 112.

Synthesis of Epifriedelanol-Hydroxy-Prolinol Succinate 113

Referring to scheme 31, Compound III (1.975 g, 2 mmol) is mixed withsuccinic anhydride (0.200 g, 2 mmol) and DMAP (0.244 g, 13 mmol) anddried in a vacuum at 40° C. overnight. The mixture is dissolved inanhydrous dichloromethane (5 mL), triethylamine (0.676 g, 0.96 mL, 6mmol) is added and the solution stirred at room temperature under argonatmosphere for 16 h. It is then diluted with dichloromethane (100 mL)and washed with ice cold aqueous citric acid (5% wt., 100 mL) and water(2×100 mL). The organic phase is dried over anhydrous sodium sulfate andconcentrated to dryness. The crude product is purified by columnchromatography to afford compound 113.

Synthesis of Epifriedelanol Immobilized Solid Support withHydroxyl-Prolinol Linker (114)

Succinate 113 (1.63 g, 1.5 mmol) is dissolved in dichloroethane (7 mL).To that solution DMAP (0.183 g, 1.5 mmol) is added.2,2′-Dithio-bis(5-nitropyridine) (0.470 g, 1 5 mmol) inacetonitrile/dichloroethane (3:1, 7 mL) is added successively. To theresulting solution triphenylphosphine (0.395 g, 1.5 mmol) inacetonitrile (3 ml) is added. The reaction mixture turned bright orangein color. The solution is agitated briefly using wrist-action shaker (5mins). Long chain alkyl amine-CPG (LCAA-CPG) (4 g, 155 μm/g) is added.The suspension was agitated for 4 h. The CPG is filtered through asintered funnel and washed with acetonitrile, dichloromethane and ethersuccessively. Unreacted amino groups are masked using aceticanhydride/pyridine. The loading capacity of the CPG is measured bytaking UV measurement.

Compound 203a:

The ester 203a was prepared according to reported procedure from theliterature (Org. Syn., 1984, 63, 183). Naproxen (201a 10.00 g, 43.427mmol, purchased from Aldrich) and 4-(Dimethylamino)pyridine (DMAP, 0.53g, 4.338 mmol, purchased from Aldrich) were dissolved in anhydrousN,N-dimethylformamide (DMF) and 1,3-diisopropylcarbodiimide (DICC, 6.8mL, 43.914 mmol, purchased from Aldrich) was added into the solution andstirred at ambient temperature for 5 minute. 6-aminohexanoic acid methylester hydrochloride (202, 10.00 g, 57.408 mmol, purchased from Fluka)and diisopropylethylamine (DIEA, 10 mL, purchased from Aldrich) wereadded into the stirring solution after 5 minute of addition of DICC andstirred overnight at ambient temperature. DMF was removed from thereaction in vacuo, the product was extracted into ethyl acetate (EtOAc,200 mL), washed successively with aqueous KHSO₄, water, sodiumbicarbonate solution and water. The organic layer was dried overanhydrous sodium sulfate (Na₂SO₄) and filtered. A white solid wasprecipitated out from the EtOAc extract by adding hexane to afford thedesired compound 203a, 11.20 g (72.14%). ¹H NMR (400 MHz, [D₆]DMSO, 25°C.): δ 7.95-7.92 (t, J(H,H)=5.2 & 5.6 Hz, 1H), 7.76-7.68 (m, 3H),7.43-7.40 (dd, J′(H,H)=1.6 and J″(H,H)=8.4 Hz, 1H), 7.25-7.24 (d,J(H,H)=2.0 Hz, 1H), 7.13-7.11 (dd, J′(H,H)=2.4 and J″(H,H)=8.8 Hz, 1H),3.84 (s, 3H), 3.70-3.65 (q, J(H,H)=6.8 and 7.2 Hz, 1H), 3.54 (s, 3H),3.00-2.97 (q, J(H,H)=6.8 Hz, 2H), 2.21-2.17 (t, 2H), 1.48-1.29 (m, 7H),1.19-1.13 (m, 2H).

Compound 204a:

Hydrolysis of the ester 203a was performed as reported earlier (Rajeevet al., 2002, 4, 4395). Compound 203a (10.80 g, 30.24 mmol) wassuspended in tetrahydrofuran—water (THF—H₂O) mixture (4:1, 40 mL) andstirred with LiOH (1.65 g, 39.32 mmol) for 4 h at ambient temperature.THF was removed from the reaction in vacuo and free acid wasprecipitated out from water by adding concentrated KHSO₄ solution,thoroughly washed with water, filtered through a sintered filter,triturated with diethyl ether and dried over P₂O₅ under vacuum overnightto obtain the acid 204a as a white solid, 10.22 g (98.4%). ¹H NMR (400MHz, [D₆]DMSO, 25° C.): δ 11.96 (bs, 1H), 7.95-7.92 (t, J(H,H)=5.37 Hz,1H), 7.77-7.68 (m, 3H), 7.43-7.41 (d, J(H,H)=8.3 Hz, 1H), 7.25-7.24 (d,J(H,H)=2.44 Hz, 1H), 7.13-7.11 (dd, J′(H,H)=1.95, 2.44 and J″(H,H)=8.79,9.27 Hz, 1H), 3.84 (s, 3H), 3.71-3.65 (q, J(H,H)=6.84, 7.33 Hz, 1H),3.02-2.97 (m, 2H), 2.13-2.09 (t, J(H,H)=7.33 Hz, 2H), 1.46-1.30 (m, 7H),1.21-1.15 (m, 2H).

Compound 205a:

Compound 204a (5.00 g, 14.57 mmol), DMAP (0.18 g, 1.47 mmol) andpentafluorophenol (3.50 g, 19.02 mmol, purchased from Aldrich) weretaken in dichloromethane (40 mL) and DCC (3.00 g, 14.54 mmol) was addedinto the solution. Reaction mixture was stirred at ambient temperaturefor 8 h. The reaction mixture was diluted to 100 mL by adding EtOAc andprecipitated DCU was removed by filtration. Combined filtrate,evaporated solvent in vacuo, and the residue was subsequently filteredthrough a column of silica gel, eluent hexane/EtOAc 4:1 to obtain amixture (7.90 g) of the desired ester 205a and excess pentafluorophenolfrom the reaction. The crude product thus obtained was directly used forproceeding experiments without further purification.

Compound 206a:

Pentafluorophenol ester 205a was stirred with serinol in the presence ofTEA to obtain compound 206a (J. Org. Chem., 1991, 56, 1713). Compound205a (4.00 g, 7.86 mmol) and serinol (1.5 g, 16.46 mmol, purchased fromAldrich) were suspended in dichloromethane (30 mL) and triethylamine(TEA, 2.3 mL, purchased from Aldrich) was added into the suspension,stirred at ambient temperature for 2 h. A white precipitate was formedduring the course of the reaction. After 2 h, the precipitate wasfiltered through a sintered filter, washed successively with excess ofdichloromethane, water and diethyl ether to afford desired product 206a(2.82 g, 86.2%). ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.95-7.92 (t,J(H,H)=5.49 Hz, 1H, exchangeable with D₂O), 7.77-7.68 (m, 3H), 7.43-7.39(m, 2H, accounted for 1H after D₂O exchange), 7.26-7.25 (d, J(H,H)=2.14Hz, 1H), 7.13-7.11 (dd, J′(H,H)=2.44 and J″(H,H)=8.85 Hz, 1H), 4.58-4.55(t, J(H,H)=5.49 Hz, 2H, exchangeable with D₂O), 3.84 (s, 3H), 3.71-3.65(m, 2H), 3.37-3.35 (t, became doublet after D₂O exchange, 4H), 3.02-2.95(m, 2H), 2.03-2.01 (t, J(H,H)=7.32, 7.63 Hz, 2H), 1.46-1.30 (m, 7H),1.20-1.12 (m, 2H).

Compound 207a:

Compound 206a was prepared by modifying reported literature procedure(Rajeev et al., Org. Lett., 2003, 5, 3005). A solid mixture of compound206a (2.50 g, 6.01 mmol) and DMAP (0.075 g, 0.61 mmol) was dried overP₂O₅ under vacuum overnight. The solid mixture was suspended inanhydrous pyridine (100 mL) under argon and heated to obtain ahomogenous solution. The temperature of the mixture was brought to roomtemperature and stirred. 4,4′-Di-O-methyltrityl chloride (2.24 g, 6.61mmol, purchased from Chem Genes Corporation) was separately dissolved in20 mL of anhydrous dichloromethane and added drop-wise into the stirringpyridine solution over a period of 45 minute under argon. Reactionmixture was further stirred overnight. Solvents were removed form thereaction mixture and the product was extracted into EtOAc (150 mL) andwashed successively with water, NaHCO₃ solution and water, dried overanhydrous Na₂SO₄ and evaporated to solid mass. Desired product waspurified by flash silica gel column chromatography: (a) eluent: 1%methylalcohol (MeOH) in dichloromethane −1.60 g of undesired bis DMTderivative (26.1%) and (b) 5% MeOH in dichloromethane −2.50 g of desiredproduct 207a (57.9%). ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.94-7.91(t, J(H,H)=5.49 Hz, 1H, exchangeable with D₂O), 7.7-7.68 (m, 3H),7.60-7.58 (d, J(H,H)=8.55 Hz, 1H, exchangeable with D₂O), 7.43-7.10 (m,12H), 6.86-6.84 (d, 4H), 4.62-4.59 (t, J(H,H)=5.18, 5.49 Hz, 1H,exchangeable with D₂O), 4.01-3.96 (m, 1H), 3.83 (s, 3H), 3.71-3.65 (m,7H), 3.44-3.42 (t, J(H,H)=5.19, 5.49 Hz, 2H), 3.03-2.87 (m, 4H),2.05-2.01 (t, J(H,H)=7.33, 7.63 Hz, 2H), 1.48-1.30 (m, 7H), 1.21-1.14(m, 2H).

Compound 208a:

The desired solid support 208a was prepared according to reportedprocedures (References for succinilation: Rajeev et al., Org. Lett.,2003, 5, 3005 and for conjugation to CPG: Kumar et al., NucleosidesNucleotides, 1996, 15, 879). A mixture of compound 207a (1.00 g, 1.39mmol), succinic anhydride (0.17 g, 1.69 mmol, purchased from Aldrich)and DMAP (0.21 g, 1.72 mmol) were suspended in 7 mL of anhydrousethylene dichloride for 24 h. Reaction mixture was diluted to 50 mL byadding dichloromethane and washed with dilute aqueous citric acidsolution (20 mL), dried over anhydrous Na₂SO₄ and evaporated to dryness.The residue obtained was further dried over P₂O₅ under vacuum to affordan almost pure but crude monosuccinate as a white solid (1.10 g, 96.5%).The product obtained was directly used for subsequent reaction withoutfurther purification. ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.94-7.91(t, J(H,H)=5.19, 5.49 Hz, 1H, exchangeable with D₂O), 7.83-7.81 (d,J(H,H)=7.94 Hz, 1H, exchangeable with D₂O), 7.76-7.68 (m, 3H), 7.42-7.10(m, 12H), 6.88-6.86 (d, 4H), 4.18-4.12 (m, 2H), 4.07-3.98 (m, 2H), 3.83(s, 3H), 3.71-3.66 (m, 7H), 3.00-2.91 (m, 4H), 2.40 (s, 4H), 2.04-2.00(t, J(H,H)=7.32 Hz, 2H), 1.44-1.22 (m, 7H), 1.19-1.15 (m, 2H).

2,2′-Dithiobis(5-nitropyridine) (0.38 g, 1.22 mmol, DTNP, purchased fromAdrich) was dissolved in a 1:1 mixture of acetonitrile and ethylenedichloride (5 mL) and added into a suspension ofnaproxen-6-aminohexanoic acid—serinol conjugate mono DMT mono succinate(1.00 g, 1.21 mmol) and DMAP (0.16 g, 1.31 mmol) in 2 mL of anhydrousacetonitrile. Triphenylphosphine (Ph₃P, 0.32 g, 1.22 mmol, purchasedfrom Aldrich) was added into the reaction mixture and shaken for 3-4minute. 5.5 g of long chain aminoalkyl controlled pore glass (CPG) with500 Å size and a loading of 112.7 μM/g (purchased from Millipore), andexcess of acetonitrile (to soak the CPG completely) were added into thereaction mixture and the suspension was shaken (agitated) for 45 minuteat ambient temperature. CPG was filtered through a sintered funnel,washed extensively with acetonitrile, dichloromethane and diethyl etherand subsequently re-suspended in pyridine-dichloromethane and treatedwith acetic anhydride in the presence of DIEA to cap unreacted aminogroups on the CPG. After 10 minute, CPG was filtered and extensivelywashed with dichloromethane, acetonitrile and diethyl ether followed bydrying under vacuum to obtain the desired CPG 208a with a loading 54.12μM/g. The loading was determined as reported in the literature (Prakashet al., J. Org. Chem., 2002, 67, 357 and references cited therein).

Compound 209a:

The phosphoramidite was prepared as reported in the literature (Rajeevet al., Org. Lett., 2003, 5, 3005 and references cited therein).Compound 207a (1.00 g, 1.39 mmol) and diisopropylammonium tetrazolide(0.12 g, 0.70 mmol) were dried over P₂O₅ vacuum overnight andsubsequently suspended in anhydrous acetonitrile (5 mL) under argonatmosphere. 2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite(0.69 mL, 2.09 mmol) was added into the suspension and stirred atambient temperature for 14 h. Solvent was removed form the reaction invacuo and residue was suspended in EtOAc (40 mL) and washed with diluteNaHCO₃ solution followed by standard work. Desired amidite 209a waspurified by flash silica gel column chromatography; eluent: 100 EtOAc,yield 0.79 g (61.8%). ³¹P NMR (161.8 MHz, CDCl₃, 25° C.): δ 146.01,145.69.

Compound 201c:

Naproxen (201, 11.25 g, 48.86 mmol), pentafluorophenol (10.00 g, 54.33mmol) and DMAP (0.60 g, 4.91 mmol) were dissolved in DMF (40 mL) andstirred at ambient temperature. 1,3-dicyclohexylcarbodiimide (DCC, 11.00g, 53.31 mmol) was added into the solution and continued stirringovernight. 1,3-dicyclohexylurea (DCU) was precipitated out during thecourse of the reaction. The precipitated DCU was filtered off, washedwith DMF, combined filtrate and removed DMF in vacuo. Oily residueobtained was filtered through a small column of silica gel, eluent 10%EtOAc in hexane to remove dissolved DCU to afford a mixture of thedesired ester 201c and excess pentafluorophenol (20.30 g). The crudeproduct thus obtained was directly used for proceeding experimentswithout further purification. ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ7.85-7.81 (m, 3H), 7.48-7.46 (dd, J′(H,H)=1.53 and J″(H,H)=8.55 Hz, 1H),7.32-7.31 (d, J(H,H)=2.44 Hz, 1H), 7.18-7.16 (dd, J′(H,H)=2.44 andJ″(H,H)=8.85 Hz, 1H), 4.47-4.44 (q, J(H,H)=7.02 Hz), 3.86 (s, 3H),1.63-1.61 (d, J(H,H)=7.34 Hz, 3H).

Compound 203b:

Ibuprofen (201b, 5.0 g, 24.23 mmol, purchased from Acros Organic),methyl 6-aminohexanoic acid monohydrochloride (202, 6.60 g, 36.33 mmol,purchased from Fluka) and DMAP (0.30 g, 2.46 mmol) were suspended indichloromethane (60 mL) in a 200 mL round bottom flask and DCC (5.00 g,24.23 mmol) was added into the suspension, stirred for 3 minute. After 3minute, 3.6 mL (25.83 mmol) of TEA was added into the reaction andcontinued stirring at ambient temperature for 18 h. Solvent and excessTEA were removed from the reaction in vacuo and residue obtained wastriturated with diethyl ether, filtered through a sintered funnel toremove DCU. Combined filtrate and evaporated on a rotary evaporator.Residue was redissolved in EtOAc (100 mL) and successively washed withKHSO₄ solution, water, NaHCO₃ solution and water followed by drying overanhydrous Na₂SO₄ and evaporation of solvent in vacuo to obtain yellowishviscous residue of compound 203b (8.0 g). The crude product thusobtained was directly used for subsequent reaction without furtherpurification. ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.86-7.84 (bt,J(H,H)=5.39, 5.00 Hz, 1H, exchangeable with D₂O), 7.19-7.03 (m, 4H),3.56 (s, 3H), 3.53-3.47 (q, J(H,H)=7.05 Hz, 1H), 3.00-2.95 (q,J(H,H)=6.64, 5.81 Hz, 2H), 2.39-2.37 (m, 2H, mixture of rotamers),2.23-2.20 (t, J(H,H)=7.45, 7.05 Hz, 2H), 1.81-1.74 (m, 1H), 1.49-1.41(m, 2H), 1.36-1.26 (m, 5H), 1.19-1.11 (m, 2H), 0.84-0.82 (m, 6H, mixtureof rotamers).

Compound 204b:

Compound 203b (8.00 g, 24.01 mmol) was stirred with LiOH (1.21 g, 28.84mmol) in THF—H₂O (4:1, 40 mL) for 4 h. Solvents were removed from thereaction mixture in vacuo and the residue was washed with concentratedKHSO₄ solution. Unlike the corresponding naproxen analogue 204a, thefree acid 204b did not precipitate out from the aqueous phase, so theaqueous phase was repeatedly extracted with EtOAc, combined extract,dried over Na₂SO₄ and evaporated in vacuo to obtain slightly yellowishviscous residue, 6.60 g (86.1%). The acid 204b thus obtained wasdirectly used for subsequent experiments without further purification.¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 11.96 (bs, 1H, exchangeable withD₂O), 7.87-7.84 (t, J(H,H)=5.39 Hz, 1H, exchangeable with D₂O),7.19-7.04 (m, 4H), 4.04-3.99 (q, J(H,H)=7.05 Hz, 1H), 3.62-3.57 (q,J(H,H)=7.05 Hz, 0.1H, minor rotamer), 3.53-3.47 (q, J(H,H)=7.05 Hz,1.9H), 3.00-2.95 (q, J(H,H)=6.22 Hz, 2H), 2.41-2.37 (m, 2H, mixture ofrotamers), 2.14-2.10 (t, J(H,H)=7.47, 7.05 Hz, 2H), 1.81-1.74 (m, 1H),1.46-1.40 (m, 2H), 1.36-1.26 (m, 5H), 1.20-1.12 (m, 2H), 0.85-0.82 (m,6H, mixture of rotamers).

Compound 206b:

Compound 204b (6.60 g, 20.676 mmol), DMAP (0.26 g, 2.128 mmol) andpentafluorophenol (5.70 g, 30.97 mmol) were dissolved in dichloromethane(60 mL) and DCC (4.27 g, 20.70 mmol) was added into the stirringsolution. The reaction mixture was allowed to stir for 8 h. PrecipitatedDCU was removed by filtration and the filtrate was evaporated to obtaina crude oil containing the desired ester 205b. The crude 205b thusobtained was stirred with serinol (3.5 g, 38.42 mmol) in dichloromethanein the presence of TEA (8 mL) for 2 h. A white precipitate was formedduring the course of the reaction, which was filtered washedsuccessively with dichloromethane, water and diethyl ether and driedover P₂O₅ to obtain 2.4 g of the product 206b. Extraction of the aqueousphase with EtOAc afforded another 1.05 g of the desired product 206b.Combined yield was 42.5%. ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ7.87-7.84 (t, J(H,H)=5.86, 5.37 Hz, 1H, exchangeable with D₂O),7.42-7.40 (d, J(H,H)=7.81 Hz, 1H, exchangeable with D₂O), 7.19-7.17 (d,J(H,H)=8.30 Hz, 2H), 7.06-7.04 (d, J(H,H)=8.30 Hz, 2H), 4.57 (bs, 2H,exchangeable with D₂O), 3.69-3.63 (m, 1H), 3.53-3.47 (q, J(H,H)=6.83 Hz,1H), 3.36-3.34 (d, J(H,H)=5.37 Hz, 4H), 3.02-2.91 (m, 2H), 2.39-2.37 (d,J(H,H)=7.34 Hz, 2H), 2.04-2.00 (t, J(H,H)=7.33 Hz, 2H), 1.81-1.75 (m,1H), 1.44-1.26 (m, 7H), 1.18-1.12 (m, 2H), 0.84-0.83 (d, J(H,H)=6.35 Hz,6H).

Compound 207b:

A solid mixture of compound 206b (3.00 g, 7.65 mmol),4,4′-dimethoxytrityl chloride (2.85 g, 8.41 mmol) and DMAP (0.20 g, 1.64mmol) was taken in a 200 mL RB and dried over P₂O₅ under vacuumovernight. Anhydrous pyridine (40 mL) was added into the mixture underargon and stirred for overnight. Pyridine was removed from the reactionand residue was suspended in EtOAc (100 mL) followed by standard workup.Desired mono DMT and bis DMT products were separated by flash silica gelcolumn chromatography, eluent: 2-3% methanol in dichloromethane, 170 g(22.3%, bis DMT derivative) and eluent: 4% methanol in dichloromethane,1.89 g (35.6%, desired mono DMT product 207b). ¹H NMR (400 MHz,[D6]DMSO, 25° C.): δ 7.83-7.80 (t, J(H,H)=5.37 Hz, 1H, exchangeable withD₂O), 7.58-7.55 (d, J(H,H)=8.79 Hz, 1H, exchangeable with D₂O),7.34-7.32 (d, J(H,H)=7.33 Hz, 2H), 7.26-7.14 (m, 9H), 7.02-7.00 (d,J(H,H)=7.81 Hz, 2H), 6.83-6.81 (d, J(H,H)=8.79 Hz, 4H), 4.58-4.56 (t,J(H,H)=5.37, 4.88 Hz, 1H, exchangeable with D₂O), 3.95-3.93 (m, 1H),3.68 (s, 6H), 3.48-3.45 (q, J(H,H)=7.34 Hz, 1H), 3.41-3.38 (t,J(H,H)=5.37 Hz, 2H), 2.96-2.84 (m, 4H), 2.34-2.33 (d, J(H,H)=7.33 Hz,2H), 2.02-1.98 (t, J(H,H)=7.33, 7.81 Hz, 2H), 1.76-1.69 (m, 1H),1.44-1.36 (m, 2H), 1.33-1.23 (m, 5H), 1.16-1.08 (m, 2H), 0.80-0.78 (d,J(H,H)=6.35 Hz, 6H). ¹³C NMR (100 MHz, [D₆]DMSO, 25° C.): δ 174.0,172.8, 158.3, 145.4, 139.9, 139.7, 136.2, 130.1, 129.2, 128.2, 128.1,127.3, 113.5, 85.5, 61.0, 55.4, 51.1, 45.1, 44.6, 35.7, 30.0, 29.1,26.3, 25.4, 22.5, 18.8.

Compound 208b:

The desired succinate (0.98 g, 85.7%) was synthesized from thecorresponding precursor 207b (1.00 g, 1.44 mmol), DMAP (0.27 g, 2.21mmol) and succinic anhydride (0.22 g, 2.20 mmol) as described for thecorresponding naproxen derivative. The succinic acid derivative waspurified by flash silica gel column chromatography, eluent: 5% methanolin dichloromethane. ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.86-7-80 (m,2H, exchangeable with D₂O), 7.34-7.32 (d, J(H,H)=7.33 Hz, 2H), 7.28-7.13(m, 9H), 7.02-7.00 (d, J(H,H)=8.30 Hz, 2H), 6.85-6.83 (d, J(H,H)=8.79Hz, 4H), 4.14-1.10 (bm, 2H), 4.02-3.98 (m, 1H), 3.68 (s, 6H), 3.50-3.44(q, J(H,H)=7.33, 6.83 Hz, 2H), 2.96-2.87 (m, 2H), 2.35-2.33 (m, 6H),2.51-2.45 (m, 7H, 2H+ DMSO-d₆), 2.01-1.96 (t, J(H,H)=7.32 Hz, 2H),1.77-1.69 (m, 1H), 1.42-1.22 (m, 7H), 1.15-1.07 (m, 2H), 0.80-0.78 (d,J(H,H)=6.35 Hz, 6H). ¹³C NMR (100 MHz, [D₆]DMSO, 25° C.): δ 174.9,174.3, 173.2, 158.5, 145.3, 139.9, 139.8, 136.0, 130.2, 129.3, 128.4,128.1, 127.4, 113.6, 85.8, 55.5, 46.1, 46.1, 45.3, 44.7, 35.6, 30.1,29.0, 26.2, 25.4, 22.6, 18.8.

The desired CPG 208b (4.50 g) with a loading capacity of 85.62 μM/g wasprepared from 0.92 g (1.16 mmol) of the ibuprofen succinate thusobtained, 2,2′-Dithiobis(5-nitropyridine) (0.37 g, 1.18 mmol), DMAP(0.15 g, 1.23 mmol), Ph₃P (0.31 g, 1.18 mmol) and long chain aminoalkylcontrolled pore glass (CPG) with 500A size and a loading of 162.5 μM/gas described for the preparation of the corresponding naproxen analogue208a.

Compound 209b:

The desired amidite 209b is prepared as described for compound 209a inExample 1.

Compound 201d:

Ibuprofen pentafluorophenol ester (201d) was prepared from ibuprofen(201b, 5.00 g, 24.23 mmol), pentafluorophenol (5.4 g, 29.02 mmol), DCC(5.00 g, 24.23 mmol) and DMAP (0.30 g, 2.46 mmol) as described for thesynthesis of pentafluorophenol ester (201c) of naproxen (201a).

Compound 210a:

Compound 4 (4.90 g, 7.35 mmol) was dissolved in ethyl acetate-methanol(4:1,16 mL) and purged with argon. To the solution was added 10%palladium on carbon (2 g, wet, Degussa type E101 NE/W). The flask waspurged with hydrogen 2 times and stirred further at room temperatureunder hydrogen at 1 atm for 2 h. The disappearance of the startingmaterial was confirmed by TLC analysis. The reaction mixture wasfiltered through a bed of Celite and washed with ethyl acetate-methanol(4:1). The combined filtrate was concentrated under reduced pressure toafford free amine. The free amine obtained was stirred with compound101c (3.1 g, 7.82 mmol) in the presence of TEA in dichloromethane (20mL) for 1 h. The reaction mixture was diluted to 50 mL and washed withaqueous sodium bicarbonate followed by standard workup. Compound 210awas obtained as a white foamy solid after flash silica gel columnchromatography, eluent: 3-4% methanol in dichloromethane, yield: 5.45 g(quant.). ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.92-7.88 (m, 1H);7.76-7.68 (m, 3H); 7.43-7.41 (d, J(H,H)=8.5 Hz, 1H); 7.31-7.08 (m, 11H);6.87-6.83 (m, 4H); 4.97 (bd, 0.7H, exchangeable with D₂O); 4.88 (bd,0.3H, exchangeable with D₂O); 4.39-4.35 (m, 0.7H); 4.29-4.26 (m, 0.3H);4.14-4.10 (m, 0.7H), 3.83-3.82 (d, J(H,H)=2 Hz, 3H, changed to multipletafter D₂O exchange); 3.71-3.65 (m, 7H); 3.54-3.50 (m, 0.7H), 3.43-3.40(m, 0.3H); 3.28-3.22 (m, 1H); 315-3.10 (m, 1H); 3.01-2.95 (m, 3H);2.12-1.80 (m, 5H); 1.42-1.04 (m, 8H).

Compound 211a:

The solid support 211a is prepared from compound 210a as described inExample 1 for the preparation of compound 208a.

Compound 212a:

The phosphoramidite 212a is prepared from compound 210a as described inExample 1 for the preparation of compound 209a.

Compound 210b:

Compound 210b is obtained from compound 4 and compound 201d as describedin Example 3 for the preparation of compound 210a.

Compound 211b:

The solid support 211b is prepared from compound 210b as described inExample 1 for the preparation of compound 208a.

Compound 212b:

The phosphoramidite 212bis prepared from compound 210b as described inExample 1 for the preparation of compound 209a.

Compound 214a:

DMT-dT-05-Amino linker (213, 1.00 g, 1.43 mmol) from Chem Genes wasstirred with cholesteryl chloroformate (0.77 g, 1.71 mmol) indichloromethane (10 mL) in the presence of TEA (1.0 mL) at ambienttemperature for 2 h. Completion of the reaction was confirmed by TCLmonitoring. The reaction mixture was diluted to 50 mL by adding moredichloromethane and washed successively with NaHCO₃ solution and waterfollowed by standard workup. Residue obtained was purified by flashsilica gel column chromatography to afford 214a (0.66 g, 37.75%). ¹H NMR(400 MHz, [D₆]DMSO, 25° C.): δ 11.61 (s, 1H, exchangeable with D₂₋O),8.01-7.98 (t, J(H,H)=5.39 Hz, 1H, exchangeable with D₂O), 7.92 (s, 1H),7.37-6.99 (m, 12H), 6.87-6.83 (m, 4H), 6.17-6.14 (t, J(H,H)=6.64 Hz,1H), 5.30 (s, 1H), 5.28-5.27 (d, J(H,H)=4.56 Hz, 1H, exchangeable withD₂O), 4.32-4.20 (m, 2H), 3.87-3.84 (m, 1H), 3.71-3.63 (m, 7H), 3.21-3.03(m, 4H), 2.95-2.88 (m, 2H), 2.33-2.13 (m, 4H), 1.97-1.73 (m, 5H),1.54-0.82 (m, 40H), 0.63 (s, 3H).

Compound 215a

Compound 214a (0.55 g, 0.495 mmol) and succinic anhydride (0.075 g,0.749 mmol) were suspended in anhydrous dichloromethane (5 mL) andstirred at ambient temperature in the presence of DMAP (0.18 g, 1.49mmol) overnight. After confirming completion of the reaction, thereaction mixture was diluted to 50 mL by adding dichloromethane andwashed with dilute citric acid solution; organic layer was dried overanhydrous Na₂SO₄ and evaporated in vacuo. Residue obtained was purifiedby flash silica gel column chromatography, eluent 6% methanol indichloromethane, to afford the corresponding succinic acid derivative(0.50 g, 83.4%). ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 12.24 (bs, 1H,exchangeable with D₂O), 11.64 (s, 1H, exchangeable with D₂O), 8.02-7.99(bm, 2H), 7.36-7.00 (m, 12H), 6.87-6.81 (m, 4H), 6.15-6.11 (t,J(H,H)=6.84 Hz, 1H), 5.30 (bs, 1H), 5.17-5.14 (bm, 1H), 4.31-4.24 (m,1H), 4.05 (bm, 1H), 3.70-3.66 (m, 8H), 3.34-3.08 (m, 6H), 2.94-2.88 (m,4H), 2.31-2.13 (m, 3H), 1.96-1.71 (bm, 5H), 1.55-0.80 (m, 40H), 0.63 (s,3H).

The succinnate thus obtained was conjugated to long chain aminoalkylcontrolled glass support (CPG) with a loading of 155 μM/g loading asdescribed in the literature by Kumar et al. (Nucleosides andNucleotides, 1996, 15, 879) to obtain the desired the desired CPG solidsupport 215a (1.70 g) with a loading of 78.42 μM/g. The loading of thesupport 215a was determined as described in the literature (Prakash etal., J. Org. Chem., 2002, 67, 357).

Compound216a

The phosphoramidite 216a is prepared from compound 214a by reacting with2-Cyanoethyl-N,N,N′, N′-tetraisopropylphosphorodiamidite in the presenceof tetrazolediisopropylammonium salt in acetonitrile according toreported procedures (Rajeev et al., Org. Lett., 2003, 5, 3005).

Compound 214b

5β-Cholanic acid (5.00 g, 13.87 mmol, purchased from Sigma),pentafluorophenol (2.81 g, 15.27 mmol, purchased from Aldrich) and DMAP(0.20 g, 1.64 mmol) were dissolved in dichloromethane andN,N′-dicyclohexycarbodiimide (DCC, 2.86 g, 13.86 mmol) was added intothe solution at ambient temperature. The reaction mixture was stirredfor 4 h. N,N′-Dicyclohexylurea was filtered off from the reaction andthe filtrate was evaporated to obtain pentafluorophenol ester of5β-cholanic acid. The ester (0.90 g, 1.708 mmol) thus obtained wasstirred with compound 213 (1.00 g, 1.431 mmol) in the presence of TEA indichloromethane (8 mL) for 2 h. The reaction was complete after 2 h asevident from TLC analysis. Reaction mixture was diluted to 50 mL byadding more dichloromethane and washed with dilute NaHCO₃ solutionfollowed by standard workup. Residue was purified by flash silica gelcolumn chromatography, eluent 3-4% methanol in dichloromethane, toafford the desired compound 214b (1.46 g, 98.04%).

¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 11.62 (bs, 1H exchangeable withD₂O); 8.03-8.00 (t, J(H,H)=5.38 Hz, 1H, exchangeable with D₂O), 7.92 (s,1H), 7.74-7.71 (t, 1H, exchangeable with D₂O); 7.37-7.02 (m, 11H),6.88-6.84 (m, 4H), 6.17-6.14 (t, J(H.H)=6.35, 6.69 Hz, 1H), 4.22-4.19(m, 1H), 3.88-3.85 (m, 1H), 3.70-3.69 (d, J(H,H)=3.91 Hz, 6H); 3.20-2.89(m, 6H), 2.33-2.27 (m, 1H), 2.18-2.12 (m, 1H), 2-08-2.00 (m, 1H),1.99-1.84 (m, 2H), 1.84-1.56 (m, 6H), 1.54-0.94 (m, 33H), 0.87-0.79 (m,7H), 0.57 (s, 3H).

¹³C NMR (100 MHz, [D₆]DMSO, 25° C.): δ 173.5, 166.1, 162.2, 158.5,149.7, 145.2, 142.9, 136.0, 135.9, 132.5, 130.1, 128.4, 128.1, 127.2,122.4, 113.6, 109.8, 107.3, 86.1, 85.9, 85.4, 70.6, 64.2, 56.5, 56.0,55.4, 55.2, 46.2, 43.5, 42.7, 40.4, 38.7, 37.5, 35.8, 35.3, 32.9, 32.1,29.5, 29.4, 28.2, 27.5, 27.1, 26.9, 26.6, 26.5, 24.4, 24.3, 21.2, 20.9,18.6, 12.2, 9.1.

Compound 215b

Compound 215b was prepared from compound 214b as described in Example 1for the synthesis of compound 215aa. Loading of the support 215b (2.7 g)prepared was determined as 81 μM/g.

Compound 216b

The phosphoramidite 216b is prepared from compound 214b by reacting with2-Cyanoethyl-N,N,N′, N′-tetraisopropylphosphorodiamidite in the presenceof tetrazolediisopropylammonium salt in acetonitrile according toreported procedures (Rajeev et al., Org. Lett., 2003, 5, 3005).

Compound 217:

N-Cbz-6-aminohexanoic acid (202, 30.31 g, 114.25 mmol, purchased fromNovabiochem), pentafluorophenol (25.00 g, 135.83 mmol) and DMAP (1.54 g,12.60 mmol) were taken in dichloromethane (100 mL) and to this DCC(26.00 g, 121.01 mmol) added slowly under stirring. During the course ofaddition, temperature of the reaction rose and dichloromethane startedboiling out, so it was cooled down to room temperature and allowed tostir overnight. Reaction mixture was diluted to 200 mL by adding diethylether and subsequently filtered through a sintered funnel to remove DCU,washed residue with diethyl ether, combined washing and evaporated todryness. The desired ester was purified by flash silica gel columnchromatography, eluent: hexane/EtOAc 2:1, yield 43.54 g (88.4%). ¹H NMR(400 MHz, [D6]DMSO, 25° C.): δ 7.36-7.23 (m, 6H), 4.99 (s, 2H),3.01-2.96 (q, J(H,H)=6.35 Hz, 2H), 2.78-2.52 (q, J(H,H)=7.33 Hz, 2H),1.69-1.61 (m, 2H), 1.47-1.29 (m, 4H).

The pentafluorophenol ester (26.00 g, 60.31 mmol) and serinol (5.00 g,54.88 mmol) were suspended in 200 mL of dichloromethane and stirred inthe presence of TEA (17 mL, 121. 97 mmol) at ambient temperatureovernight. A thick white precipitate was formed during the course of thereaction. The reaction mixture was diluted to 200 mL by adding diethylether, triturated and filtered. The precipitate was thoroughly washedwith diethyl ether and dried under vacuum over P₂O₅ to obtain 16.51 g(81.0%) of the desired compound 217 as a white solid. ¹H NMR (400 MHz,[D₆]DMSO, 25° C.): δ 7.44-7.42 (d, J(H,H)=7.81 Hz, 1H, exchangeable withD₂O), 7.37-7.27 (m, 5H), 7.24-7.20 (t, J(H,H)=5.86, 5.37 Hz, 1H,exchangeable with D₂O), 4.99 (s, 2H), 4.58-4.55 (t, J(H,H)=5.37 Hz, 2H,exchangeable with D₂O), 3.70-3.65 (m, 1H), 3.37-3.34 (t, J(H,H)=5.86,3.37 Hz, changed to doublet after D₂O exchange, J(H,H, after D₂Oexchange)=5.37 Hz, 4H), 2.98-2.92 (q, J(H,H)=6.84, 6.35 Hz, 2H),2.06-2.02 (t, J(H,H)=7.33 Hz, 2H), 1.49-1.33 (m, 4H), 1.24-1.16 (m, 2H).

Compound 218:

Compound 217 (14.10 g, 41.66 mmol) and DMAP 0.60 g, 4.91 mmol) weretaken in a 200 mL RB and dried under vacuum over P₂O₅. The solid mixturethen suspended in 50 mL of anhydrous pyridine under argon.4,4-Dimethoxytrityl chloride (15.5 g, 44.27 mmol) was separatelydissolved in 40 mL of anhydrous dichloromethane and added into thestirring pyridine solution under argon. The reaction mixture was allowedto stir at ambient temperature overnight. Solvents were removed from thereaction mixture and residue was extracted into EtOAC (200 mL), washedwith NaHCO₃ solution followed by standard workup. The desired product218 was purified by flash silica gel column chromatopgraphy, eluent:hexane/EtOAc 3:2, 8.62 g (28.0%, bis DMT derivative) and 3-4% MeOH inchloroform, 15.28 g (57.3%, desired mono DMT derivative 218). ¹H NMR(400 MHz, [D₆]DMSO, 25° C.): δ 7.63-7.60 (d, J(H,H)=8.79 Hz, 1H,exchangeable with D₂O), 7.38-7.17 (m, 15H, accounted for 14H after D₂Oexchange), 6.87-6.84 (d, J(H,H)=8.79 Hz, 4H), 4.98 (s, 2H), 4.62-4.59(t, J(H,H)=5.37 Hz, 1H, exchangeable with D₂O), 4.00-3.95 (m, 1H), 3.72(s, 6H), 3.46-3.41 (t, J(H,H)=5.37 Hz, 2H), 3.00-2.87 (m, 4H), 2.08-2.04(t, J(H,H)=7.33 Hz, 2H) 1.50-1.33 (m, 4H), 1.25-1.16 (m, 2H).

Compound 219:

Compound 218 (12.91 g, 20.16 mmol) in anhydrous pyridine (50 mL) wasstirred with TBDMS-Cl (4.60 g, 30.52 mmol) in the presence of imidazole(6.30 g, 92.54 mmol) at ambient temperature for 6 h. After 6 h pyridinewas removed in vacuo and the product was extracted into ethyl acetate(100 mL), washed with sodium bicarbonate solution followed by standardworkup. The residue was purified by flash silica gel columnchromatography, eluent: 2-3% methanol in dichloromethane to affordcompound 219 (15.10 g, 99.3%). ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ7.65-7.63 (bd, J(H,H)=8.30 Hz, 1H, exchangeable with D₂O); 7.38-7.17 (m,14H); 6.86-6.84 (d, J(H,H)=8.79 Hz, 4H); 5.00 (s, 2H); 4.01-3.96 (m,1H); 3.71 (s, 6H); 3.58-3.52 (m, 2H), 3.04-2.99 (m, 1H); 2.98-2.89 (m,3H); 2.09-2.05 (t, 2H); 1.50-1.43 (m, 2H); 1.42-1.38 (m, 2H); 124-1.19(m, 2H); 0.75 (s, 9H); −0.05 (s, 3H); −0.06 (s, 3H).

Compound 220:

Compound 219 (7.05 g, 9.33 mmol) and ammonium formate (3.00 g, 47.573)were suspended in 40 mL of methanol/ethyl acetate (1:2) and to this Pd—C(10%, 0.70 g) was added at ambient temperature. The suspension wasinitially warmed by blowing hot air and subsequently stirred at ambienttemperature for 4 h. Completion of the reaction was monitored by TLC andafter 4 h, the reaction mixture was filtered over a celite column,washed residue with methanol/ethyl acetate (1:2), combined filtrate andevaporated to dryness. Residue obtained was extracted into ethyl acetate(100 mL) and washed with aqueous sodium bicarbonate and water. Organiclayer was dried over anhydrous Na₂SO₄ and evaporate to obtain the freeamine. ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.72-7.61 (m, 1.5H);7.43-7.06 (m, 10H); 6.86-6.84 (d, J(H,H)=8.79 Hz, 4H); 4.20-3.97 (bm,1H); 3.71 (s, 6H); 3.59-3.52 (bm, 2H); 3.07-3.00 (m, 2H); 2.93-2.88 (m,2H); 2.72-2.98 (t, 1H); 2.10-2.05 (m, 2H); 1.53-1.44 (m, 4H), 1.28-1.22(m, 2H), 0.75 (s, 9H); −0.05 (s, 3H); −0.07 (s, 3H).

The free amine (2.0 g, 3.22 mmol) was stirred with biotin-NHS ester (1.0g, 2.93 mmol, purchased from Sigma) in the presence of triethylamine inDMF for 4 h. Progress of the reaction was monitored by TLC. Removed DMFin vacuo and the product was extracted into ethyl acetate (50 mL) andwashed with water followed by standard workup. Compound 220 was purifiedby flash silica gel column chromatography; eluent: 5% methanol indichloromethane, yield: 1.43 g (57.7%). ¹H NMR (400 MHz, [D₆]DMSO, 25°C.): δ 7.73-7.70 (t, J(H,H)=5.38 Hz, 1H, exchangeable with D₂O);7.66-7.63 (d, J(H,H)=8.79 Hz, 1H, exchangeable with D₂O); 7.38-7.18 (m,9H); 6.86-6.84 (d, J(H,H)=8.30 Hz, 4H); 6.42-6.35 (d, J(H,H)=27.35 Hz,2H, changes to 6 5.62-5.61 with J(H,H)=0.98 Hz after D₂O exchange);4.29-4.26 (m, 1H), 4.12-4.08 (m, 1H), 4.00-3.98 (m, 1H), 3.72 (s, 6H),3.57-3.53 (m, 2H), 3.08-2.77 (m, 6H); 2.57-2.54 (d, J(H,H)=12.70 Hz,1H), 2.10-2.06 (t, J(H,H)=8.79, 5.86 Hz, 2H); 2.04-2.00 (t, J(H,H)=7.32Hz, 2H), 1.61-1.19 (m, 12H); 0.75 (s, 9H), −0.05 (s, 3H); −0.06 (s, 3H).

Compound 221:

Compound 220 (1.3 g, 1.54 mmol) was stirred with 4-tert-butylbenzoylchloride (1 mL, 5.08 mmol) in the presence of DMAP (0.02 g, 0.163 mmol)in anhydrous pyridine (5 mL) under argon atmosphere for 4 h. Excess of4-tert-butylbenzoyl chloride was quenched by adding methanol andsubsequently removed pyridine and methanol in vacuo. The product wasextracted into ethyl acetate (50 mL) and washed with aqueous sodiumbicarbonate followed by standard workup. Residue obtained was subjectedto flash column chromatography to afford compound 221 (0.64 g, 41.4%).¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.72-7.69 (t, J(H,H)=5.37 Hz, 1H,exchangeable with D₂O); 7.64-7.62 (d, J(H,H)=8.79 Hz, 1H, exchangeablewith D₂O); 7.53-7.17 (m, 14H); 6.83-6.81 (d, J(H,H)=8.79 Hz, 4H);5.11-5.10 (d, J(H,H)=1.95 Hz, 2H); 4.01-3.98 (m, 1H); 3.71 (s, 6H);3.61-3.55 (m, 3H); 3.18-3.15 (m, 1H); 3.03-2.88 (m, 4H); 2.07-1.99 (m,4H); 1.62-1.58 (m, 1H); 1.47-1.19 (m, 22H); 0.75 (s, 9H), −0.05 (s, 3H);−0.07 (s, 3H).

¹³C NMR (100 MHz, [D₆]DMSO, 25° C.): δ 173.0, 172.9, 172.8, 172.7,170.5, 170.0, 158.4, 155.5, 155.4, 152.2, 145.4, 136.2, 136.1, 132.0,131.6, 130.2, 129.3, 129.1, 128.2, 128.1, 127.1, 125.2, 113.5, 85.6,62.4, 62.2, 60.0, 59.9, 55.5, 55.2, 54.2, 50.8, 35.8, 35.1, 31.2, 31.1,29.2, 29.0, 28.7, 26.4, 26.1, 25.6, 18.2, −5.1, −5.0.

Compound 222:

A solution of compound 221 (0.64 g, 0.64 mmol) in anhydrous THF (5 mL)was stirred with TEA.3HF (purchased from Aldrich, 1 mL) in the presenceof anhydrous TEA (5 mL) at ambient temperature for 24. Solvents wereremoved from the reaction mixture under vacuum and the product wasextracted into ethyl acetate, washed with aqueous sodium bicarbonatefollowed by standard workup. Flash silica gel column chromatography(eluent: 5% methanol in dichloromethane) of the residue affordedcompound 222 (0.54 g, 95%). ¹H NMR (400 MHz,

[D6]DMSO, 25° C.): δ 7.91 (bs, 1H, exchangeable with D₂O); 7.73 (bt, 1H,exchangeable with D₂O); 7.63-7.61 (d, J(H,H)=8.79 Hz, 1H, exchangeablewith D₂O); 7.39-7.19 (m, 13H); 6.87-6.84 (d, J(H,H)=8.79 Hz, 4H);5.05-5.02 (m, 1H), 4.62-4.60 (t, J(H,H)=5.37 Hz, 1H, exchangeable withD₂O); 4.21-4.17 (m, 1H); 4.00-3.96 (m, 1H); 3.72 (s, 6H); 3.45-3.42 (t,J(H,H)=5.37 Hz, 2H, changed to a doublet after D₂O exchange); 3.28-3.22(m, 1H); 3.04-2.84 (m, 6H), 2.10-1.94 (m, 4H); 1.71-1.62 (m, 1H);1.57-1.18 (m, 22H).

¹³C NMR (100 MHz, [D₆]DMSO, 25° C.): δ 172.8, 172.7, 169.5, 158.3,155.8, 154.2, 145.4, 136.2, 132.8, 130.1, 128.8, 128.1, 128.0, 127.0,124.6, 113.4, 85.4, 63.0, 62.1, 61.0, 57.6, 55.4, 55.2, 55.1, 51.1,57.6, 55.4, 55.2, 55.1, 38.7, 37.7, 35.8, 35.6, 35.0, 31.3, 29.2, 28.6,28.2, 26.4, 25.6, 25.5.

Compound 223:

The phosphoramidite 223 is prepared from compound 222 by reacting with2-Cyanoethyl-N,N,N′, N′-tetraisopropylphosphorodiamidite in the presenceof tetrazolediisopropylammonium salt in acetonitrile according toreported procedures (Rajeev et al., Org. Lett., 2003, 5, 3005).

Compound 224:

Compound 222 (0.50 g, 0.56 mmol) was stirred with succinic anhydride(0.115 g, 1.15 mmol) in the presence of DMAP (0.21 g, 1.72 mmol) inanhydrous ethylene dichloride under argon at 55° C. for 3 h. Thereaction mixture was diluted to 20 mL by adding dichloromethane andwashed with cold 10% citric acid solution, dried over anhydrous sodiumsulfate and evaporated to dryness. The acid formed was purified by flashsilica gel column chromatography (eluent: 10% methanol indichloromethane); yield: 0.50 g (89.9%). ¹H NMR (400 MHz, [D6]DMSO, 25°C.): δ 12.12 (s, 1H, exchangeable with D₂O); 7.91 (bs, 1H, exchangeablewith D₂O); 7.86-7.84 (bd, 1H, exchangeable with D₂O); 7.74-7.72 (bd, 1H,exchangeable with D₂O);

7.52-7.16 (m, 13H); 6.88-6.86 (d, J(H,H)=8.79 Hz, 4H); 5.06-5.02 (m,1H), 5.02-4.95 (m, 0.4H); 4.82-4.76 (m, 0.6H); 4.27-3.89 (m, 5H);3.79-3.76 (m, 0.4H); 3.72 (s, 6H); 3.54-3.48 (m, 0.6H); 3.41-2.81 (m,9H, accounted for 8H after D₂O exchange); 2.52 (bm, 4H); 2.07-1.94 (bm,4H); 1.71-1.62 (m, 1H); 1.54-1.13 (m, 22H).

¹³C NMR (100 MHz, [D₆]DMSO, 25° C.): δ 174.5, 174.1, 173.3, 173.2,172.9, 172.7, 169.8, 158.6, 156.1, 155.9, 154.5, 145.3, 136.0, 132.9,130.2, 129.3, 129.0, 128.5, 128.1, 127.4, 125.4, 124.8, 113.7, 85.8,63.5, 62.3, 57.8, 55.6, 55.4, 55.2, 57.8, 55.6, 55.4, 55.2, 48.0, 37.9,35.8, 35.2, 31.4, 31.4, 29.2, 29.1, 29.0, 28.8, 28.4, 26.4, 25.8, 25.6.

Compound 224 (2.0 with 56.15 iM/g loading) was prepared from the acid(0.45 g, 0.45 mmol), DMAP (0.068 g, 0.56 mmol), triphenylphosphine(0.135 mg, 0.51 mmol) and 2,2′-Dithiobis(5-nitropyridine) (DTNP, 0.16 g,0.52 mmol) as described for the synthesis of compound 208a.

Compound 225a:

Compound 4a (12.09 g, 18.23 mmol) was stirred with TBDMS-C1 (4.00 g,26.54 mmol) in the presence of imidazole (5.42 g, 79.61 mmol) inanhydrous pyridine (60 mm) overnight. After removing pyridine, theproduct was extracted into ethyl acetate (150 mL), washed with aqueoussodium bicarbonate, followed by standard workup. Residue obtained wassubjected to flash silica gel column chromatography using 1% methanol indichloromethane as eluent to afford compound 225a. ¹H NMR (500 MHz,[D₆]DMSO, 25° C.): δ 7.33-7.13 (bm, 15H, accounted for 14H after D₂Oexchange); 6.87-6.82 (bm, 4H); 5.01 (s, 0.2H, rotamer minor); 4.99 (s,1.8H, rotamer major), 4.68-4.64 (m, 0.72 H, major rotamer); 4.14-4.07(bm, 1H), 3.72 (s, 7H), 3.38-3.36 (m, 0.6H, rotamer minor); 3.26-3.21(m, 1.4H, rotamer major); 3.08-3.07 (m, 0.3H, rotamer, minor); 2.99-2.89(m, 2.7H, rotamer, major); 2.22-2.12 (m, 2H), 2.04-1.78 (m, 2H);1.48-1.23 (m, 6H), 0.84, 0.82 (s, 9H, rotamers major and minor); 0.05(d, J(H,H)=1.5 Hz, 4.3H, rotamer major); 0.03-0.02 (d, J(H,H)=5.5 Hz,1.7H).

Compound 226a

Compound 225a (12.22 g, 15.67 mmol) was hydrogenated at 1 atm over 10%Pd—C (1.12 g, wet Degussa type E101 NE/W) in ethyl acetate/methanol(4:1) for 4 h as described for the synthesis of compound 210a. The freeamine obtained was stirred with biotin-NHS ester (5.42 g, 15.87 mmol,purchased from ChemGenes Corporation Wilmington, Mass.) in the presenceof TEA for 6 h in dichloromethane/methanol (9:1, 100 mL). Solvents wereremoved from the reaction mixture and the product was extracted intodichloromethane (400 mL), washed with aqueous sodium bicarbonatefollowed by standard workup. The crude product thus obtained was usedfor next reaction without further purification or characterization.

Compound 227a:

The crude 226a and DMAP (0.31 g, 2.57 mmol) were taken in anhydrouspyridine and stirred over an ice bath. To the stirring solution,4-tert-butylbenzoyl chloride (5.0 mL, 25.42 mmol) was added drop-wiseover ten minute. After the addition, the reaction nmixture was broughtto room temperature over 2 h and continued stirring overnight. Afterquenching excess 4-tert-butylbenzoyl chloride by adding methanol,solvents were removed from the reaction mixture and the product wasextracted into ethyl acetate (300 mL), washed with aqueous sodiumbicarbonate followed by standard workup. The desired product 227a waspurified by flash silica gel column chromatography using dichloromethanecontaining 4-6% of methanol as eluent. Yield: 9.53 g (58.9%). ¹H NMR(500 MHz, [D₆]DMSO, 25° C.): δ 7.90 (m, 1H, exchangeable with D₂O);7.73-7.72 (bm, 1H, exchangeable with D₂O); 7.39 (s, 4H); 7.32-7.16 (m,9H), 6.88-6.86 (m, 4H); 5.05-5.02 (m, 1H); 4.68-4.64 (m, 0.7H, rotamer,major); 4.57-4.53 (m, 0.3H, rotamer, minor); 4.20-4.17 (m, 1H),4.13-4.08 (m, 1H); 3.72 (s, 6H), 3.38-3.20 (m, 3H); 3.08-2.81 (m, 5H);2.24-1.77 (m, 6H); 1.68-0.98 (m, 23H); 0.84-0.81 (m, 9H), 0.05-0.02 (m,6H).

Compound 228a

Compound 227a (6.43 g, 6. 22 mmol) was taken in a 250 ml RB and to this3 mL of anhydrous TEA and 20 mL of 1M TBAF in anhydrous THF (purchasedfrom Aldrich) were added under argon and stirred at ambient temperaturefor 4 h. Progress of the reaction was monitored by TLC, and after 4 h,THF was removed in vacuo. Residue was extracted into ethyl acetate (100mL), washed with aqueous sodium bicarbonate followed by standard workup.Compound 228a was obtained as a white foamy solid after flash silica gelcolumn chromatography (eluent: 5-6% methanol in dichloromethane), yield:5.51 g (96.3%). ¹H NMR (500 MHz, [D₆]DMSO, 25° C.): δ 7.90 (bs, 1H,exchangeable with D₂O); 7.76-7.74 (m, 1H, exchangeable with D₂O); 7.39(s, 4H), 7.32-7.15 (m, 9H); 6.88-6.84 (m, 4H); 5.08-5.01 (m, 1H),4.98-4.97 (d, 0.7H, exchangeable with D₂O); 4.89-4.88 (d, 0.3H,exchangeable with D₂O), 4.40-4.38 (m, 0.85H), 4.30-4.27 (m, 0.3H);4.21-4.18 (m, 0.85H); 4.17-4.05 (m, 1H); 3.72 (s, 6H); 3.58-3.52 (m,0.85H), 3.44-3.38 (m, 0.5H); 3.34-3.28 (m, 0.85H); 3.27-3.22 (m, 1H),3.19-3.14 (m, 0.8H); 3.04-2.81 (m, 5H), 2.21-1.77 (m, 6H), 1.68-1.20 (m,22H).

Compound 229a:

After drying over P₂O₅ under vacuum, compound 228a (1.49 g, 1.62 mmol)was taken in anhydrous dichloroethane (10 mL) under argon and stirred atambient temperature. To the solution anhydrous TEA (0.70 mL, 5.02 mmol)and N,N-diisopropylamino 13-cyanoethylphosphonamidic chloride (0.80 mL,3.38 mmol, purchased from ChemGenes Corporation, Wilmington, Mass.) wereadded and stirred for 3 h. After completion of the reaction, solvent andexcess TEA were removed under vacuum and the product was extracted intoethyl acetate, washed with aqueous sodium bicarbonate solution followedby standard workup. Phosphoramidite 229a was purified by flash silicagel column chromatography using ethyl acetate as eluent, yield: 0.48 g(26.4%). ³¹P NMR (162 MHz, [D₆]DMSO, 25° C.): δ 149.02 (major); 148.90(minor); 148.62 (minor); 148.02 (major).

Compound 230a:

Compound 228a (1.55 g, 1.68 mmol) and DMAP (1.0 g, 8.18 mmol) were takenin anhydrous dichloroethane (5 mL) and stirred at ambient temperature.Succinic anhydride (0.30 g, 2.99 mmol) was added into the stirringsolution and the stirring was continued overnight. The succinatederivative was obtained as a gray white solid (0.61 g, 35.5%) afterworkup and purification as described for the preparation of compound224. ¹H NMR (500 MHz, [D₆]DMSO, 25° C.): δ 12.15 (s, 1H, exchangeablewith D₂O); 7.75-7.58 (bm, 1.4H, exchangeable with D₂O); 7.54-7.47 (m,4H); 7.32-7.16 (m, 9H); 6.89-6.81 (m, 4H); 5.38-5.33 (m, 0.7H);5.28-5.20 (m, 0.3H); 5.06-5.00 (m, 0.3H); 4.99-4.95 (m, 0.7H); 4.82-4.77(m, 1H); 4.28-4.22 (m, 0.25H); 4.21-4.15 (m, 0.75H); 3.80-3.60 (m, 7H),3.57-3.42 (m, 2H); 3.22-2.80 (m, 7H); 2.28-1.90 (m, 6H), 1.58-1.41 (m,24H).

¹³C NMR (100 MHz, [D₆]DMSO, 25° C.): δ 174.3, 174.2, 173.2, 173.1,172.7, 171.8, 170.3, 158.7, 158.5, 158.4, 155.9, 152.5, 145.4, 145.1,140.6, 136.3, 136.1, 135.9, 131.9, 130.2, 129.4, 129.2, 128.4, 128.1,127.3, 125.3, 113.8, 113.7, 113.3, 85.9, 73.4, 72.5, 63.6, 59.9, 55.6,55.2, 52.8, 5.9, 35.8, 35.3, 35.0, 34.6, 33.5, 31.6, 31.4, 31.3, 29.3,29.1, 28.6, 28.5, 28.1, 26.5, 25.5, 24.6.

The solid support 230a (2.05 g with a loading of 66.88 μM/g) wasobtained from the succinate (0.57 g, 0.55 mmol), DMAP (0.085 g, 0.69mmol), triphenylphosphine (0.15 g, 0.57 mmol), DTNP (0.18 g, 0.58 mmol)and lca-CPG (155 μM/g loading with a mean pore diameter of 484 Å,purchased from Millipore) as described for the preparation of compound208a.

Compound 225b:

Compound 225b is prepared from compound 4b as described for thesynthesis of compound 225a.

Compound 226b:

The desired compound is obtained from compound 225b as described for thepreparation of compound 226a from compound 225a.

Compound 227b:

The desired compound is obtained from compound 226b as described for thepreparation of compound 227a from compound 226a.

Compound 228b:

Compound 228b is prepared from compound 227b as described for thesynthesis of compound 228a from compound 227a.

Compound 229b:

The desired compound is obtained from compound 228b as described for thepreparation of compound 229a from compound 228a.

Compound 230b:

Compound 230b is prepared from compound 228b as described for thesynthesis of compound 230a from compound 228a.

Compound 232a:

Tert-Butyl ester (231a) of 6-hydroxyhexanoic acid is prepared asreported in the literature (Larock and Leach, J. Org. Chem., 1984, 49,2144). Compound 231a is reacted with N-hydroxyphthalimide underMitsunobu conditions to obtain compound 232a (as reported by Katajistoet. al., Bioconjugate Chem., 2004, 15, 890.

Compound 233a:

Compound 232a is treated with trifluoroacetic acid to obtain compound233a.

Compound 234a:

The free acid 233a is stirred with N-hydroxysuccinimide and DCC in thepresence of DAMP in DMF for 30 min and subsequently compound 38 is addedinto the reaction mixture to obtain the desired compound 234a.

Compound 235a:

Compound 234a is treated with hydrazine.hydrate in pyridine andsubsequently with 5-cholesten-3-one (purchased from Aldrich) to obtaincompound 235a.

Compound 236a:

The phosphoramidite 236a is prepared from 235a as described for thecompound 7 from compound 6 using2-cyanoethyl-N,N,N′,N″-tetraisopropylphosphorodiamidite as thephosphitylation agent.

Compound 237a:

The solid support 237a is obtiend from compound 235a as described forthe synthesis of compound 208a.

Compound 238a:

Compound 235a is treated with sodium cyanoborohydride to reduce the C≡Ndouble bond. The crude product of the sodium cyanoborohydride reactionis subsequently treated with ethyl trifluoroacetate in the presence ofTEA in dichloromethane to obtain compound 238a.

Compound 239a:

The phosphoramidite 239a is prepared from 238a as described for thecompound 7 from compound 6 using2-cyanoethyl-N,N,N′,N″-tetraisopropylphosphorodiamidite as thephosphitylation agent.

Compound 240a:

The solid support 240a is obtiend from compound 238a as described forthe synthesis of compound 208a.

Compound 232b:

Compound 231b is prepared as reported in the literature by Noguchi etal. (Tetrahedron, 1995, 51, 10531). Compound 231b is converted to 232bas described for the preparation of compound 232a.

Compound 236b:

The desired prosphoramidite is obtiend from compound 232b in four stepsas described for the prepaprtion of compound 236a from 232a.

Compound 237b:

The CPG support is obtiend from compound 235b as described for thesynthesis of compound 208a from 207a.

Compound 238b:

Compound 238b is prepared from compound 235b as described for thepreparation of compounc 238a from 235a.

Compound 239b:

The phosphoramidite 239b is prepared from 238b as described for thecompound 7 from compound 6 using2-cyanoethyl-N,N,N′,N″-tetraisopropylphosphorodiamidite as thephosphitylation agent.

Compound 240b:

The solid support 240b is obtiend from compound 238b as described forthe synthesis of compound 208a.

Compound 241a:

Naproxen pentafluorophenol ester (1.3 g, 3.28 mmol) was added into asolution of compound 213 (1.5 g, 2.14 mmol, purchased from ChemGenesCorporation, Wilmington, Mass.) and TEA (4.6 mL, 33.0 mmol) and stirredovernight. Solvent and excess TEA were removed from the reaction invacuo and the product was extracted into ethyl acetate (80 mL), whasedwth aqueous sodium bicarbonate solution followed by standard workup.Flash silica gel column chromatography of the residue usingdichloromethane containing 4% methanol as eluent yielded 0.85 g (43.5%)of compound 241a as a grayish white solid. ¹H NMR (400 MHz, [D₆]DMSO,25° C.): δ 11.62 (s, 1H, exchangeable with D₂O); 7.99-7.91 (bm, 3H),7.76-7.68 (m, 3H); 7.43-7.01 (m, 13H); 6.87-6.84 (m, 4H); 6.17-6.14 (t,J′(H,H)=6.41 and J″(H,H)=6.71 Hz, 1H), 5.28-5.27 (d, J(H,H)=4.88 Hz, 1H,exchangeable with D₂O); 4.23-4.19 (m, 1H); 3.88-3.81 (bm, 4H), 3.69-3.68(bm, 8H); 3.20-2.97 (m, 6H); 2.34-2.27 (m, 1H); 2.19-2.13 (m, 1H);1.38-1.17 (bm, 11H).

Compound 242a:

Compound 241a (0.65 g, 0.71 mmol) and DMAP (0.13 g, 1.06 mmol) weretaken in dichloroethane (5 mL) in an RB and stirred at ambienttemperature. Succinic anhydride (0.11 g, 1.09 mmol) was added into thestirring solution and the stirring was continued for 24 h. Progress ofthe reaction was monitored by TLC and after 24 h, the reaction mixturewas diluted to 50 mL by adding ethyl acetate. Organc layer was washedwith cold dilute citric acid solution follwed by water. Organic layerwas dried over anhydrous sodium sulfate and evaporated in vacuo toobtain the corresponding succinic acid derivative (0.67 g, 92.8%, crudeyield) of compound 241a.

The succinate (0.51 g, 0.50 mmol) thus obtiend was converted to compound242a (2.8 g, with a loading of 11.6 μM/g) by coupling to lca-CPG (2.8 gwith initial loading of 112.7 μM/g with mean pore size 505 Å, purchasedfrom Millipore) using triphenylphosphine (0.132 g, 0.503 mmol), DAMP(0.07 g, 0.57 mmol) and 2,2′-dithiobis(5-nitropyridine) (DTNP, 0.156 g,0.50 mmol) as coupling agents as described for the synthesis of compound208a.

Compound 243a:

The phosphoramidite 243a is prepared from 241a as described for thecompound 7 from compound 6 using2-cyanoethyl-N,N,N′,N″-tetraisopropylphosphorodiamidite as thephosphitylation agent.

Example 16 Oligonucleotide Synthesis, Purification and Analysis

Synthesis:

The Oligonucleotide molecules were synthesized on a 394 ABI machineusing the standard 93 step cycle written by the manufacturer withmodifications to a few wait steps as described below. The solid supportwas available in house and the monomers were RNA phosphoramidites withfast protecting groups (5′-O-dimethoxytritylN6-phenoxyacetyl-2′-O-t-butyldimethylsilyladenosine-3′-O—N,N′-di isopropyl-cyano ethylpho sphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-O-t-butyldimethylsilylcytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-p-isopropylphenoxyacetyl-2′-O-t-butyldimethylsilylguanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, and5′-O-dimethoxytrityl-2′-O-t-butyldimethylsilyluridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramiditefrom Pierce Nucleic Acids Technologies. All 2′-O-Me amidites werereceived from Glen Research. All amidites were used at a concentrationof 0.15M in acetonitrile (CH₃CN) and a coupling time of 12-15 min. Theactivator was 5-(ethylthio)-1H-tetrazole (0.25M), for the PO-oxidationIodine/Water/Pyridine was used and for PS-oxidation, 2% Beaucage reagent(Iyer et al., J. Am. Chem. Soc., 1990, 112, 1253) in anhydrousacetonitrile was used. The sulphurization time was about 6 min.

Deprotection-I (Nucleobase Deprotection)

After completion of synthesis the support was transferred to a screw capvial (VWR Cat #20170-229) or screw caps RNase free microfuge tube. Theoligonucleotide was cleaved from the support with simultaneousdeprotection of base and phosphate groups with 1.0 mL of a mixture ofethanolic ammonia [ammonia:ethanol (3:1)] for 15 h at 55° C. The vialwas cooled briefly on ice and then the ethanolic ammonia mixture wastransferred to a new microfuge tube. The CPG was washed with 2×0.1 mLportions of RNase free deionised water. Combined washings, cooled over adry ice bath for 10 min and subsequently dried in speed vac.

Deprotection-II for RNA oligonucleotides (Removal of 2′ TBDMS group)

The white residue obtained was resuspended in 400 μl of triethylamine,triethylamine trihydrofluoride (TEA.3HF) and NMP (4:3:7) and heated at50° C. for overnight to remove the tert-butyldimethylsilyl (TBDMS)groups at the 2′ position (Wincott et al., Nucleic Acids Res., 1995, 23,2677). The reaction was then quenched with 400 μl ofisopropoxytrimethylsilane (iPrOMe₃Si, purchased from Aldrich) andfurther incubated on the heating block leaving the caps open for 10 min;(This causes the volatile isopropxytrimethylsilylfluoride adduct tovaporize). The residual quenching reagent was removed by drying in aspeed vac. Added 1.5 ml of 3% triethylamine in diethyl ether andpelleted by centrifuging. The supernatant was pipetted out withoutdisturbing the pellet and the pellet was dried in speed vac. The crudeRNA was obtained as a white fluffy material in the microfuge tube.

Quantitation of Crude Oligomer or Raw Analysis

Samples were dissolved in RNase free deionied water (1.0 mL) andquantitated as follows: Blanking was first performed with water alone (1mL) 20 μL of sample and 980 μL of water were mixed well in a microfugetube, transferred to cuvette and absorbance reading obtained at 260 nm.The crude material is dried down and stored at −20° C.

5. Purification of Oligomers: PAGE Purification

PAGE purification of oligomers synthesized was performed as reported bySambrook et al. (Molecular Cloning: a Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).The 12% denaturing gel was prepared for purification of unmodified andmodified oligoribonucleotides. Took 120 mL Concentrate+105 mL Diluents+25 mL Buffer (National Diagnostics) then added 50 μL TEMED and 1 5 mL10% APS. Pour the gel and leave it for ½ h to polymerize. Suspended theRNA in 20 μL water and 80 μL formamide. Load the gel tracking dye onleft lane followed by the sample slowly on to the gel. Run the gel on1×TBE buffer at 36 W for 4-6 h. Once run is completed, Transfer the gelon to preparative TLC plates and see under UV light. Cut the bands. Soakand crushed in Water. Leave in shaker for overnight. Remove the eluent,Dry in speed vac.

HPLC Analysis and Purification

Analysis was performed on an Agilent 1100 series HPLC using a Dionex4×250 mm DNAPak column. Buffer A was 1 mM EDTA, 25 mM Tris pH9, 50 mMNaClO₄, 20% MeCN. Buffer B was 1 mM EDTA, 25 mM Tris pH 9, 0.4 M NaClO₄,20% MeCN. Separation was performed on a O-65% B segmented gradient withbuffers and column heated to 65° C.

Materials were purified on an ÅKTA Explorer equipped with a columnpacked with TSKgel Q 5PW (Tosoh Biosciences). Buffer A was 1 mM EDTA, 25mM Tris pH 9. Buffer B was 1 mM EDTA, 25 mM Tris pH 9, 0.4 M NaClO₄.Buffers were heated by a 4 kW buffer heater set at 65° C., giving acolumn outlet temperature of 45° C. The solution containing the crudematerial was diluted 4-6 fold and loaded onto the column and eluted witha segmented gradient from O-60% B. Appropriate fractions were pooled

Desalting of Purified Oligomer

The purified dry oligomer was then desalted using Sephadex G-25 M(Amersham Biosciences). The cartridge was conditioned with 10 mL ofRNase free deionised water thrice. Finally the purified oligomer wasdissolved in 2.5 mL RNasefree water and passed through the cartridgewith very slow drop wise elution. The salt free oligomer was eluted with3.5 mL of RNase free water directly into a screw cap vial. Allolgonucleotides were finally analyzed by LC-MS and capillary gelelectrophoresis.

TABLE 6  List of ligand oligonucleotides (sense and antisense strand).Found Sequence Cal Mass Mass CGE ID Sequence amu amu (%) 100 5′CUU ACG CUG AGU ACU UCG A dTdT 3′ 6606.00 6606.45 99.25 101 5′UCG AAG UAC UCA GCG UAA G dT dT 3′ 6696.32 6693.0  89.0  102 5′CUU ACG CUG AGU ACU UCG A dTdT L₁3′ 7084.19 7084.58 96.90 103 5′UCG AAG UAC UCA GCG UAA G dT dT L₁ 3′ 7170.29 7170.89 92.00 104 5′CUU ACG CUG AGU ACU UCG A dT dr L₁ 3′ 7100.19 7099.12 92.99 105 5′UCG AAG UAC UCA GCG UAA G dT dT* L₁ 3′ 7157.29 7156.2  89.00 106 5′G*G*U*G*U*A U G G C U U C A A C C* C* 7293.00 7237.06 97.50U* U*_(2′OMe)U*_(2′OMe) L₁ 3′ 107 5′A* G* G* G* U U G A A G C C A U* A C* 7362.73 7338.4  96.00A* C* C* U*_(2′OMe)U_(2′OMe) L₁ 3′ 108 5′CUU ACG CUG AGU ACU UCG A dT dT L₂ 3′ 7064.96 7064.91 90.0% 109 5′UCG AAG UAC UCA GCG UAA G dT dT L₂ 3′ 7154.00 7153.2  90.72 110 5′UCG AAG UAC UCA GCG UAA G UU L₂ 3′ 7153.98 7151.13 92.20 111 5′L₁CUU ACG CUG AGU ACU UCG A dTdT 3′ 112 5′L₁UCG AAG UAC UCA GCG UAA G dT dT 3′ 113 5′L₂CUU ACG CUG AGU ACU UCG A dTdT 3′ 114 5′L₂UCG AAG UAC UCA GCG UAA G dT dT 3′ 115 5′CUU ACG CUG AGU ACU UCG A dTdTL₃3′ 116 5′UCG AAG UAC UCA GCG UAA G dT dTL₃ 3′ 117 5′L₃ CUU ACG CUG AGU ACU UCG A dTdT 3′ 118 5′L₃ UCG AAG UAC UCA GCG UAA G dT dT 3′ 119 5′CUU ACG CUG AGU ACU UCG A dTdT L₄ 3′ 120 5′UCG AAG UAC UCA GCG UAA G dT dT L₄ 3′ 121 5′L₄ CUU ACG CUG AGU ACU UCG A dTdT 3′ 122 5′L₄ UCG AAG UAC UCA GCG UAA G dT dT 3′ 123 5′CUU ACG CUG AGU ACU UCG A dT dT L₅ 3′ 124 5′UCG AAG UAC UCA GCG UAA G dT dT L₅ 3′ 125 5′L₅CUU ACG CUG AGU ACU UCG A dT dT 3′ 126 5′L₅UCG AAG UAC UCA GCG UAA G dT dT 3′ 127 5′CUU ACG CUG AGU ACU UCG A dT dT L₆ 3′ 128 5′UCG AAG UAC UCA GCG UAA G dT dT L₆ 3′ 129 5′L₆CUU ACG CUG AGU ACU UCG A dT dT 3′ 130 5′L₆UCG AAG UAC UCA GCG UAA G dT dT 3′ 131 5′CUU ACG CUG AGU ACU UCG A dT dT L₇ 3′ 132 5′UCG AAG UAC UCA GCG UAA G dT dT L₇ 3′ 133 5′L₇CUU ACG CUG AGU ACU UCG A dT dT 3′ 134 5′L₇UCG AAG UAC UCA GCG UAA G dT dT 3′ 135 5′CUU ACG CUG AGU ACU UCG A dT dT L₉ 3′ 136 5′UCG AAG UAC UCA GCG UAA G dT dT L₉ 3′ 137 5′L₈CUU ACG CUG AGU ACU UCG A dT dT 3′ 138 5′L₈UCG AAG UAC UCA GCG UAA G dT dT 3′ 139 5′CUU ACG CUG AGU ACU UCG A dT dT L₉ 3′ 140 5′UCG AAG UAC UCA GCG UAA G dT dT L₉ 3′ 141 5′L₉CUU ACG CUG AGU ACU UCG A dT dT 3′ 142 5′L₉UCG AAG UAC UCA GCG UAA G dT dT 3′ 143 5′CUU ACG CUG AGU ACU UCG A dT dT L₁₀ 3′ 144 5′UCG AAG UAC UCA GCG UAA G dT dT L₁₀ 3′ 145 5′L₁₀CUU ACG CUG AGU ACU UCG A dT dT 3′ 146 5′L₁₀UCG AAG UAC UCA GCG UAA G dT dT 3′ 147 5′CUU ACG CUG AGU ACU UCG A dT dT L₁₁ 3′ 148 5′UCG AAG UAC UCA GCG UAA G dT dT L₁₁ 3′ 149 5′L₁₁CUU ACG CUG AGU ACU UCG A dT dT 3′ 150 5′L₁₁UCG AAG UAC UCA GCG UAA G dT dT 3′ 151 5′CUU ACG CUG AGU ACU UCG A dT dT L₁₂ 3′ 152 5′UCG AAG UAC UCA GCG UAA G dT dT L₁₂ 3′ 153 5′L₁₂CUU ACG CUG AGU ACU UCG A dT dT 3′ 154 5′L₁₂UCG AAG UAC UCA GCG UAA G dT dT 3′ 155 5′CUU ACG CUG AGU ACU UCG A dT dT L₁₃ 3′ 156 5′UCG AAG UAC UCA GCG UAA G dT dT L₁₃ 3′ 157 5′L₁₃CUU ACG CUG AGU ACU UCG A dT dT 3′ 158 5′L₁₃UCG AAG UAC UCA GCG UAA G dT dT 3′ 159 5′CUU ACG CUG AGU ACU UCG A dT dT L₁₄ 3′ 160 5′UCG AAG UAC UCA GCG UAA G dT dT L₁₄ 3′ 161 5′L₁₄CUU ACG CUG AGU ACU UCG A dT dT 3′ 162 5′L₁₄UCG AAG UAC UCA GCG UAA G dT dT 3′ 163 5′CUU ACG CUG AGU ACU UCG A dT dT L₁₅ 3′ 164 5′UCG AAG UAC UCA GCG UAA G dT dT L₁₅ 3′ 165 5′L₁₅CUU ACG CUG AGU ACU UCG A dT dT 3′ 166 5′L₁₅UCG AAG UAC UCA GCG UAA G dT dT 3′ 167 5′CUU ACG CUG AGU ACU UCG A dT dT L₁₆ 3′ 168 5′UCG AAG UAC UCA GCG UAA G dT dT L₁₆ 3′ 169 5′L₁₆CUU ACG CUG AGU ACU UCG A dT dT 3′ 170 5′L₁₆UCG AAG UAC UCA GCG UAA G dT dT 3′ 171 5′CUU ACG CUG AGU ACU UCG A dT dT L₁₇ 3′ 172 5′UCG AAG UAC UCA GCG UAA G dT dT L₁₇ 3′ 173 5′L₁₇CUU ACG CUG AGU ACU UCG A dT dT 3′ 174 5′L₁₇UCG AAG UAC UCA GCG UAA G dT dT 3′ L₁ = Naproxen 6-aminohexanoic acidwith Serinol linker L₂ = Ibuprofen 6-aminohexanoic acid with Serinollinker L₃ = Cholesterol 6-aminohexanoic acid withtrans-4-hydroxy-L-prolinol linker L₄ = Cholesterol 6-aminohexanoic acidwith serinol linker L₆ = Cholesterol with trans-4-hydroxy-L-prolinollinker containing cationic tert-amine moiety L₇ = Thiocholesterol withtrans-4-hydroxy-L-prolinol linker L₈ = Cholesterol 6-aminohexanoic acidwith 3-hydroxy-4-(hydorxy)methylpyrrolidine linker L₈ = Biotin6-aminohexanoic acid with trans-4-hydroxy-L-prolinol linker L₉ = Biotin6-aminohexanoic acid with serinol linker L₁₀ = Biotin 12-aminododecanoicacid with trans-4-hydroxy-L-prolinol linker L₁₀ = ω-aminocaproyl withtrans-4-hydroxy-L-prolinol linker L₁₁ = ω-aminododecyl withtrans-4-hydroxy-L-prolinol linker L₁₂ = Vitamin E 6-aminohexanoic acidwith trans-4-hydroxy-L-prolinol linker L₁₃ = Dialkylglyceride6-aminohexanoic acid with trans-4-hydroxy-L-prolinol linker L₁₄ =Naproxen 6-aminohexanoic acid with trans-4-hydroxy-L-prolinol linker L₁₅= N,N-Dimethyl 6-aminohexanoic acid with trans-4-hydroxy-L-prolinollinker L₁₆ = N,N-Dimethyl 12-aminododecanoic acid withtrans-4-hydroxy-L-prolinol linker L₁₇ = Nadixic 6-aminohexanoic acidwith trans-4-hydroxy-L-prolinol linker *= PS

Example 17 siRNA Modifications Enhanced Duplex Stability

Radiolabel Method for Monitoring Serum Stability of siRNA Duplexes:

siRNA duplexes were prepared at a stock concentration of 1 μM in whicheither the sense (S) or antisense strand (AS) contained a trace amountof 5′-³²P labeled material (e.g. ³²P—S/AS and S/³²P-AS). The presence ofthe end-labeled sense or antisense strand allowed for monitoring of theindividual strand within the context of the siRNA duplex. Therefore, twoduplex preparations were made for each siRNA sequence tested. siRNAduplexes were incubated in 90% human serum at a final concentration of100 nM duplex. Samples were removed and quenched in a stop mix atappropriate times. For a typical time course, 10 seconds, 15 minutes, 30minutes, 1 hour, 2 hours and 4 hours time points were taken. Sampleswere analyzed by denaturing polyacrylamide gel electrophoresis alongwith a control sample (4 hour buffer-alone incubation) and a partialalkaline hydrolysis ladder of the labeled sense or antisense strand as amarker. The gel was imaged using a Fuji phosphorimager to detect thefull length sense and antisense strands along with any degradationfragments that were generated by serum nucleases during incubation.

Since there is the possibility of losing the 5′ phosphate label due tophosphatase activity in the serum, an alternative to 5′ end labeling isto place an internal ³²P or ³³P label within either the sense orantisense strand. This labeling method is much more laborious than 5′end labeling and currently we have no evidence that dephosphorylationoccurs during serum incubation.

A series of chemical modifications that fall into the followingcategories; backbone modification, sugar modification, nucleobasemodification and 3′ conjugate, were tested and showed enhanced serumstability as compared to a unmodified siRNA duplex. A description ofeach modification, its location within the siRNA duplex, and the serumstability data follows.

Serum Stability of Unmodified Parent Duplex:

The unmodified parent duplex, AL-DUP-1000, was used to establish theserum stability baseline for evaluating the effect of chemicalmodifications on nuclease resistance.

AL-DUP-1000 SEQ ID NO: 54 5′-CUUACGCUGAGUACUUCGAdTdT-3′ ALN-SEQ-1000SEQ ID NO: 61 3′ dTdTGAAUGCGACUCAUGAAGCU-5′ ALN-SEQ-1001

AL-DUP-1000 was subjected to the serum stability assay to evaluate itsinherent nuclease resistance and to define its degradation pattern (FIG.21). Denaturing gel electrophoresis was used analyze AL-DUP-1000 in ahuman serum stability assay. An siRNA duplex containing 5′ end-labeledsense RNA (*s/as) and a duplex containing 5′ end-labeled antisense RNA(as/s*) were each incubated in 90% human serum and time points wereassayed at 10 seconds, 5 min, 15 min, 30 min, 1 hour, 2 hours and 4hours. The control was a 4 hour time point for siRNA duplex incubated inPBS buffer alone, OH— was the partial alkaline hydrolysis marker. Thisunmodified duplex was observed to be degraded by both 3′-5′ exonucleasesand endonucleases (FIG. 21).

Cleavage of the 3′ end of both the sense and antisense strands by 3′-5′exonucleases occurs within the first 5 minutes of incubation resultingin the loss of the 3′ terminal dT residues (top vertical lines in s*/asand s/as* panels of FIG. 21). In addition to exonuclease degradation,both strands were cleaved by endonucleases. There was a majorendonuclease site at position sixteen of the antisense strand (bottomvertical lines in s*/as and s/as* panels of FIG. 21) that appears asearly as 10 seconds. Very little full length sense or antisense strandwas remaining after 1 hour in human serum. Chemical modifications wereintroduced within the context of the parent duplex to evaluate theireffect on nuclease resistance. These chemical modifications fall withinone of the following classes: backbone modification, sugar modification,nucleobase modification, cationic modification and conjugate.

Backbone Modifications Enhanced Nuclease Resistance:

Specific phophodiester linkages of the siRNA duplex were replaced byeither phosphorothioate or methylphosphonate and their stability wasevaluated in the human serum stability assay. Table 7 contains thesequences of the duplexes tested. Substitution of the phosphodiesterlinkage at the 3′ end of both the sense and antisense strands inhibitedexonucleolytic degradation of the 3′ overhangs (FIGS. 22A and 22B) ascompared to the unmodified parent duplex (refer to FIG. 21). Full lengthstarting material was present for four hours for both the sense andantisense strands. The endonucleolytic cleavage pattern seen in theunmodified duplex was unchanged. Similar results were obtained forduplexes that contained additional phosphorothioates at their 3′ ends(data not shown). The placement of phosphorothioates at theendonucleolytic cleavage sites (duplexes 1419, 1420 and 1421) did notinhibit endonucleolytic cleavage at these sites (data not shown). Insummary, a single phosphorothioate or methylphosphonate between the two3′ terminal nucleotides was sufficient to protect the 3′ ends fromexonuclease degradation. Additional phosphorothioates at the 3′ endsappear to enhance this effect, which may be necessary for long termexposure to serum nucleases.

TABLE 7  siRNA duplexes containing backbone modifications. AlnylamAlnylam Duplex Duplex Sequence Sequence AL-DUP-5′-CUUACGCUGAGUACUUCGAdT*dT-3′ AL-SEQ- 1393 10263′-dT*dTGAAUGCGACUCAUGAAGCU-5′ AL-SEQ- 1027 AL-DUP-5′-CUUACGCUGAGUACUUCGA*dT*dT-3′ AL-SEQ- 1394 10283′-dT*dT*GAAUGCGACUCAUGAAGCU-5′ AL-SEQ- 1029 AL-DUP-5′-CUUACGCUGAGUACUUCG*A*dT*dT-3′ AL-SEQ- 1395 10303′-dT*dT*G*AAUGCGACUCAUGAAGCU-5′ AL-SEQ- 1031 AL-DUP-5′-CUUACGCUGAGUACUUC*G*A*dT*dT-3′ AL-SEQ- 1396 10323′-dT*dT*G*A*AUGCGACUCAUGAAGCU-5′ AL-SEQ- 1033 AL-DUP-5′-CUUACGCUGAGU*ACUUCGAdTdT-3′ AL-SEQ- 1419 21823′-dTdTGAAUGCGACUCA*UGAAGCU-5′ AL-SEQ- 2184 AL-DUP-5′-CUU*ACGCUGAGU*ACUUCGAdTdT-3′ AL-SEQ- 1420 21833′-dTdTGAA*UGCGACUCA*UGAAGCU-5′ AL-SEQ- 2185 AL-DUP-5′-CUU*ACGCUGAGU*ACUUCGAdT*dT-3′ AL-SEQ- 1421 21863′-dT*dTGAA*UGCGACUCA*UGAAGCU-5′ AL-SEQ- 2188 AL-DUP-5′-CUUACGCUGAGUACUUCGAdTmpdT-3′ AL-SEQ- 1329 10383′-dTmpdTGAAUGCGACUCAUGAAGCU-5′ AL-SEQ- 1039 (*= phosphorothioate, mp =methylphosphonate)SEQ ID NOs 62-77, respectively.

Sugar Modifications Enhanced Nuclease Resistance:

The effect of replacing the 2′OH with 2′OMe was evaluated at the sitesof endonucleolytic cleavage as well as at the 3′ ends of the siRNAduplex. The duplexes tested in the human serum stability assay are shownin Table 2. Some of these duplexes also contained phosphorothioatelinkages to evaluate whether the combination of the two modificationsenhance nuclease resistance more significantly. Substitution of theterminal dT residues with 2′OMe-U (AL-DUP-1027) reduced 3′-5′exonuclease degradation slightly over the unmodified parent duplex (datanot shown); however, the extent of exonuclease protection by 2′OMe-U wasfar less than that achieved by placing a

TABLE 8  siRNA duplexes containing 2′OMe Substitutions Alnylam AlnylamDuplex Duplex Sequence Sequence AL-DUP- 5′-CUUACGCUGAGUACUUCGAUU-3′AL-SEQ-1006 1027 3′-UUGAAUGCGACUCAUGAAGCU-5′ AL-SEQ-1007 AL-OUP-5′-C*UUACGCUGAGUACUUCGAU*U-3′ AL-SEQ-1008 10363′-U*UGAAUGCGACUCAUGAAGC*U-5′ AL-SEQ-1009 AL-DUP-5′-C*UUACGCUGAGUACUUCGAU*U,-3′ AL-SEQ-gggg 13ff3′-U*UGAAUGCGACUCAUGAAGC*U-5′ AL-SEQ-hhhh AL-DUP-5′-C*UUACGCUGAGUACUUCGAU*U-3′ AL-SEQ-1162 13633′-U*UGAAUGCGACUCAUGAAGC*U-5′ AL-SEQ-1163 (U = 2′OMe-uridine, * =phosphorothioate)

SEQ ID NOs 78-85, respectively.

phosphorothioate between the two terminal dT residues (see FIG. 22A).Addition of a single phosphorothioate between the two terminal2′OMe-uridine residues effectively inhibited 3′-5′ exonucleolyticcleavage as seen in FIG. 23 for duplexes AL-DUP-1036, AL-DUP-13ff, andAL-DUP-1363. 2′OMe substitution on its own was much more effective atprotecting from endonucleolytic cleavage when placed at the internalcleavage sites. The parent duplex was cleaved 3′ of U at two UpA siteswithin the duplex. Both strands are cleaved due to the symmetry of thisdinucleotide repeat and mapping data was used to confirm the sites ofcleavage (data not shown). Placement of 2′OMe at the strongendonucleolytic site ((FIG. 23, star in s/*as gel, AL-DUP-13ff) resultedin inhibition of cleavage at this site. The second, weakerendonucleolytic site (FIG. 23, black star in *s/as), however, wasslightly enhanced when the strong site was protected with 2′OMe (FIG.23, compare AL-DUP-13ff to AL-DUP-1036). Protection of both sites with2′OMe (AL-DUP-1363) resulted in reduced endonucleolytic cleavage at bothsites (FIG. 23). The inhibitory effect of the 2′OMe substitution isconsistent with the mechanism of endonucleolytic cleavage, whichrequires the 2′OH as a nucleophile in the cleavage reaction. 2′OMemodification will also be an effective means to protect the 3′ overhangof single overhang siRNA duplexes where the 3′ overhang is composed ofribonucleotides. In this situation, 2′OMe substitution can be used toblock the possible loss of the terminal two nucleotides byendonucleolytic cleavage and phosphorothioate can be used to protectfrom exonuclease degradation.

Cationic Modifications Enhanced Nuclease Resistance:

The effect of three different cationic chemical modifications onnuclease resistance was tested and compared to the parent unmodifiedduplex. The structures of the three cationic modifications tested areshown below.

TABLE 9  siRNA duplexes containing cationic substances Alnylam AlnylamDuplex Duplex Sequence Sequence AL-DUP- 5′-CUUACGCUGAGUACUUCGAdTaadT-3′AL-SEQ- 10aa 1017 3′-aadTdTGAAUGCGACUCAUGAAGCU-5′ AL-SEQ- 1018 AL-DUP-5′-CUUACGCUGAGUACUUCGAaadTaadT-3′ AL-SEQ- 10bb 10153′-aadTaadTGAAUGCGACUCAUGAAGCU-5′ AL-SEQ- 1016 AL-DUP-5′-CUUACGCUGAGUACUUCGAdTdTAbP-3′ AL-SEQ- lccc dddd3′-AbPdTdTGAAUGCGACUCAUGAAGCU-5′ AL-SEQ- eeee AL-DUP-5′-C*UaaUACGCUGAGUACUUCGAU*U-3′ AL-SEQ- 1403 20803′-U*UGAAaaUGCGACUCAUGAAGC*U-5′ AL-SEQ- 2081 AL-DUP- 5′C*UaaUACGCUGAGaaUACUUCGAU*U-3′ AL-SEQ- 1406 20823′-U*UGAAaaUGCGACUCAaaUGAAGC*U-5′ AL-SEQ- 2083 (aadT = alkylamine-dt,abP = abasic pyrrolidine cationic, aaU = allylamine-U, *=phosphorothioate, U = 2′OMe-U)SEQ ID NOs 86-95, respectively.The sequences of the duplexes assayed in the human serum stability assayare shown in Table 9.

Both alkylamino-dT and abasic pyrrolidine cationic modifications wereplaced at the 3′ terminal overhang to evaluate their effect on 3′-5′exonuclease degradation. Allylamino-uridines were placed at the internalendonucleolytic cleavage sites to evaluate their ability to inhibitendonucleolytic cleavage. As seen in FIG. 24, replacing the 3′ terminaldT residue with a single alkylamino-dT efficiently inhibited 3′-5′exonucleolytic degradation (FIG. 24, AL-DUP-10aa, left gel image).Replacement of both dT residues in the overhang with alkylamino-dTresulted in a similar extent of inhibition (data not shown). Addition ofan abasic pyrrolidine cationic modification at the 3′ terminus of eachstrand also protected against exonucleolytic degradation (FIG. 24,middle gel image). Both the alkylamino-dT and abasic pyrrolidinemodifications protected from 3′-5′ exonucleolytic cleavage up to 23hours (data not shown). Placement of allylamino-U at the internalcleavage sites inhibited endonucleolytic cleavage as shown in FIG. 24for duplex AL-DUP-1403. The ends of this duplex were stabilized fromexonucleolytic degradation by 2′OMe-U and phosphorothioate substitutionsin order to separate the two different cleavage events. Endonucleolyticcleavage was inhibited at both internal cleavage sites by allylamino-Usubstitution for AL-DUP-1406 (data not shown).

3′ Conjugates Enhanced Nuclease Resistance:

Conjugation of naproxen and ibuprofen to the 3′ end of the siRNA weretested for their ability to inhibit 3′-5′ exonucleolytic degradation.The structure of naproxen is shown in below:

Table 10 lists the siRNAs that were tested in the human serum stabilityassay. Conjugation of either naproxen or ibuprofen to the 3′ endinhibited exonucleolytic degradation.

FIG. 18 shows the serum stability data for the naproxen modified duplex(AL-DUP-1069) and similar results were obtained for AL-DUP1413.Presumably the conjugates inhibit exonucleolytic cleavage by stericallyblocking the exonuclease from binding to the 3′ end of the siRNA duplex.Similar data was also obtained for AL-DUP-1069 in pooled mouse serum.

TABLE 10  siRNA duplexes containing 3′ conjugates Alnylam Alnylam DuplexDuplex Sequence Sequence AL-DUP- 5′-CUUACGCUGAGUACUUCGAdTdTNap-3′ 10693′-NapdTdTGAAUGCGACUCAUGAAGCU-5′ AL-DUP-5′-CUUACGCUGAGUACUUCGAdTdTIbu-3′ 1413 3′-NapdTdTGAAUGCGACUCAUGAAGCU-5′(Nap = Naproxen, Ibu = Ibuprofen)

SEQ ID NOs 96-99, respectively.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A modified RNA agent comprising a sense strand and an antisense strand, wherein one or more ribose replacement modification subunit (RRMS) comprising a ligand is incorporated into at least one of said strands, and wherein the RRMS is a cyclic carrier.
 2. The modified RNA agent of claim 1, wherein the cyclic carrier is a carbocyclic ring system or a heterocyclic ring system.
 3. The modified RNA agent of claim 1, wherein the cyclic carrier is selected from the group consisting of hydroxyproline, piperidine, morpholine, piperazine, and decalin.
 4. The modified RNA agent of claim 1, wherein the RRMS is incorporated into the sense strand.
 5. The modified RNA agent of claim 4, wherein the RRMS is incorporated into the 3′ end of the sense strand.
 6. The modified RNA agent of claim 5, wherein the RRMS is placed within 1, 2, or 3 positions of the 3′ end of the sense strand.
 7. The modified RNA agent of claim 1, wherein the RRMS is incorporated into the antisense strand.
 8. The modified RNA agent of claim 7, wherein the RRMS is incorporated into the 3′ end of the antisense strand.
 9. The modified RNA agent of claim 1, wherein the sense strand and the antisense strand are independently 17 to 25 nucleotides in length.
 10. The modified RNA agent of claim 1, wherein the modified RNA agent includes a duplex region between 17 and 23 pairs in length.
 11. The modified RNA agent of claim 1, wherein the modified RNA agent includes at least one 3′ overhang of 2-3 nucleotides in length.
 12. The modified RNA agent of claim 1, wherein the ligand alters the distribution, targeting or lifetime of a RNA agent into which it is incorporated.
 13. The modified RNA agent of claim 1, wherein the ligand is selected from the group consisting of a folic acid radical; a steroid radical; a carbohydrate radical; a vitamin A radical; a vitamin E radical; and a vitamin K radical.
 14. The modified RNA agent of claim 1, wherein the ligand is a cholesterol radical.
 15. The modified RNA agent of claim 1, wherein the ligand is a carbohydrate radical.
 16. The modified RNA agent of claim 1, wherein the ligand is a targeting group selected from the group consisting of mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, and an RGD peptide or RGD peptide mimetic.
 17. The modified RNA agent of claim 1, wherein at least two RRMS subunits are incorporated into at least one of said strands.
 18. The modified RNA agent of claim 17, wherein at least two RRMS subunits are incorporated into the sense strand.
 19. The modified RNA agent of claim 17, wherein at least two RRMS subunits are incorporated into the antisense strand. 